Abstract The analysis of oligopeptides in samples of food tissues and body fluids attracts considerable attention The complexity of such samples requires efficient sample preparation (ie concentration and cleanup) procedures to remove interfering endogenous compounds and inorganic or organic salts The methods of sample pretreatment that enable effective and selective isolation andor preconcentration of oligopeptides from complex sample matrices have been reviewed In each case examples of application were presented and discussed taking into account selectivity enrichment method automation cleanup and environmental aspects of the developed methods
Keywords Body fluids Oligopeptides Sample preparation
Introduction
Although the discovery of oligopeptides at the turn of nineteenth and twentieth centuries as products of the protein hydrolysis initially did not draw too much attention the significant role of this group of compounds in the functioning of all living organisms has since become well known Nowadays it is generally accepted that the existence of oligopeptides in the human body is indispensable for the proper functioning of numerous internal biological systems [1 2] Oligopeptides include hormones neurotransmitters metabolic regulators antibiotics antioxidants antalgic drugs or even toxins [2–7] Moreover
A Poliwoda (*) P P Wieczorek Institute of Chemistry Opole University Pl Kopernika 11
45–040 Opole Poland
these compounds play an important role in preservation of the rheological properties of food [8 9] Oligopeptides as a final product of food hydrolysis (mostly milk cheese and wine) are known to act as very good antioxidants anticoagulants and blood pressure reducers [10–12] The importance of synthetic oligopeptides has also grown in recent years and these molecules have found wide application in medicine including the therapy of some incurable diseases [13–17]
The widespread occurrence and biological activity of oligopeptides brought about the necessity to determine their presence and concentration in various types of samples Usually the analysis has to be done on very complex matrices such as body fluids (blood plasma serum cerebrospinal fluid or urine) as well as protein hydrolysates of food These samples contain a large variety of different endogenous compounds so most analytical systems are unable to handle them simultaneously Almost without exception oligopeptides are present in such samples at low concentrations together with interfering endogenous large substances (proteins and lipids) that make them difficult to isolate and determine with good accuracy Furthermore the interfering substances can often be confused with the peptides For that reason even with the access of advanced separation and detection techniques it is rather difficult to analyze oligopeptide samples without an adequate sample preparation step The application of specific procedures for isolation purification and preconcentration of analytes is commonly required Due to the complexity of matrices it is often not sufficient to use one sample preparation technique so several methods have to be applied to meet the requirements of proper analysis
Thus far not many papers have been published dealing with techniques for sample preparation in analysis of oligopeptides from complex sample matrices [7 18 19]
There are only a few reports describing sample cleanup procedures applied for the analysis of foodderived peptides [20–22] and these have mainly dealt with specific analytical techniques (eg capillary electrophoresis and HPLC) or a specific group of peptides generally with long amino acid chains [23–26] Only a few communications have described details about applications of only one specific sample pretreatment technique in peptide analysis [21 27 28] The data on sample preparation techniques used for the analysis of small peptides (oligopeptides) are very limited This review is therefore focused on commonly used sample cleanup methods that have been applied for isolation and preconcentration of oligopeptides from various complex matrices In each case examples of applications will be considered and discussed taking into account the selectivity enrichment method automation cleanup and environmental aspects of the developed methods
Analysis on crude samples
The perfect situation for each analyst is to have a method that enables a direct analysis without the need for any additional steps Unfortunately such a situation happens very rarely and is limited to only a few analytical detection techniques like fluorescence detection and immunoassays (RIA ELISA) For example HPLC analysis with fluorescence detection was applied to the determination of a synthetic octapeptide used as an antiviral drug in rabbit plasma samples [16] This compound contains the naturally fluorescent tyrosine residue so no derivatization was required The analyzed plasma samples were only diluted with water before direct injection The sensitivity of this method was about 5 μgl or even lower However the analysis involving naturally present fluorescent components is not very common in the case of oligopeptides Usually for the fluorescence detection technique the use of a fluorescent derivatization agent is essential [29–31]
A few methods that utilized a variety of fluorescent derivatization schemes for the detection of oligopeptides by capillary electrophoresis have been described [30 31] As a simple example the method for the determination of glutathione (GSH) in must and wine can be mentioned [30] In these experiments besides the use of a 20minlong derivatization procedure the must and wine samples were only centrifuged prior to analysis The detection limit was 20 μgl However the method concerned only the analysis of samples of white wine which contains smaller amounts of various interferents that may give a false response and decrease the recovery of analytes
Immunological methods allow detection of peptides at very low concentrations and with high specificity for a given analyte Unfortunately the use of immunological
detection in the case of peptide analysis is limited only to compounds against which specific antibodies have been generated For example the direct injection of complex sample matrices (plasma) without any manipulation was used in the analysis of larger polypeptides (28–32 amino acids) against which specific antibodies have been produced [32–34] However the specificity of immunological assays may be questionable especially if they are not combined with HPLC For instance the presence of proteins and salts in biological samples often results in RIA assay inaccuracy In addition problems with crossreactivity of a specific antibody with compounds that are structurally similar to the analytes forces the use of some sample cleanup steps Such a problem was observed during the determination of some opioid peptides and substance P in human cerebrospinal fluid (CFS) [35] The greater amount of albumin in the CSF sample interfered in the assay and caused an overestimation of neuropeptide levels when the RIA was done without a prior separation step Therefore it was impossible to determine these oligopeptides directly To overcome this problem a liquid–liquid extraction method was used with success
To conclude in the case of oligopeptide analysis there are only few examples of direct injection procedures of complex sample matrices into analytical instruments Usually the complexity of various interfering compounds makes oligopeptide analysis impossible Hence the use of a suitable sample pretreatment procedure is required For instance the presence of some colorants preservatives and antioxidants in food samples (like in red wine samples) limited the effectiveness of direct fluorescence analysis Furthermore the concomitant derivatization of plasma interferents in oligopeptide analysis caused decreased sensitivity Thus in order to avoid these limitations additional methods for removal of endogenous compounds and oligopeptide preconcentration are almost always necessary Taking into account that usually complex samples are analyzed (eg blood urine or food) a direct analysis could be done in only a few cases when the content of analytes of interest in the sample was high
Preliminary sample cleanup
Several cleanup methods are currently available when samples containing oligopeptides need to be characterized and these furthermore depend on the type of sample to be analyzed (liquid or solid) however the need to use many operating steps during the sample pretreatment procedure increases the probability of sample contamination Therefore the possibility of applying simple preliminary pretreatment methods like homogenization precipitation and filtration for preseparation of the analyte of interest
from a complex matrix may be a more desirable approach The application of a simple preliminary pretreatment step like homogenization andor precipitation can sometimes be sufficient
Homogenization
Homogenization of tissue samples such as a brain liver kidney or food (meat and cheese) is the first step of sample pretreatment commonly used before protein precipitation enzymatic digestions or extraction techniques Sometimes some of the large proteins are removed by the application of filtration after sample homogenization Several methods for homogenization of samples containing oligopeptides have been described in the literature [9 19 36–45]
Generally the biological tissues containing oligopeptides are homogenized in solution of saline or buffers [19 38 39 41] It has been noticed that the stability of analytes depends on the pH of applied buffer as well as type and concentration of organic acid For example the superiority of trifluoroacetic acid over trichloroacetic acid has been proven [46] When homogenization and sample storage were performed at 4 °C structural changes of the peptides were not observed [47] The use of buffers and a mixture of organic solvents helps in isolation of very hydrophobic peptides [40] Complete homogenization of tissues was usually achieved in 10–30 s which was an additional advantage of this technique In all cases the application of homogenization helped to prepare a uniform mixture and disperse minute fragments of tissue Sometimes homogenization and centrifugation were sufficient for effective sample cleanup as was accomplished in an investigation of angiotensin II formation in the human heart interstitium [41] The obtained heart homogenates (NaCl at pH 74 and a temperature of 4 °C for 1 min) were centrifuged and the supernatant was then evaporated to dryness dissolved in 01 trifluoroacetic acid solution and injected directly into an HPLC instrument without any problems
Regarding the analysis of oligopeptides from food samples homogenization methods were mostly applied on cheese samples [36 42 44] In general water and aqueous buffers were the most commonly used for extraction of oligopeptides from cheese No influence of homogenization temperature on the level of extractable peptides has been observed The recommended ratio of water to cheese was 21 which afforded high values of analytes recovery even over 90 when the homogenization process was performed in two repetitions In many cases the sample obtained after homogenization was merely filtered and directly injected
Homogenization is therefore rather a supplementary and necessary preliminary step used for isolation of oligopeptides from solid and semisoft samples For illustration
homogenization in water for only 15 s was applied for quantitative determination of the two antihypertensive tripeptides ValProPro and IleProPro from various types of cheese [42] Owing to the high content of the two bioactive peptides in the studied samples no further cleanup and preconcentration stage was required After the homogenization the sample was centrifuged and the filtered supernatant was directly analyzed by HPLCMS Homogenization was therefore a fast and sometimes very efficient method of sample pretreatment for oligopeptides analysis However its application is limited and depends mainly on the oligopeptide content which has to be high to enable determination Usually after homogenization the application of some other sample pretreatment techniques is required in order to obtain a proper sample cleanup andor oligopeptide preconcentration
The preliminary process of homogenization with distilled water and hydrolysis was not enough to determine the small antioxidant oligopeptides from a protein hydrolysate of mackerel [45] Therefore the protein hydrolysate was additionally homogenized with cold 10 trichloroacetic acid a typical precipitation agent However the complexity of the sample required the use of a liquid–liquid extraction step to increase the analyte recovery as will be discussed later A similar situation occurred in the case of isolation of some regulatory peptides from porcine brain [39] In this method after homogenization (with icecold acetic acid ascorbate and ethylenediaminetetraacetic acid) the samples were centrifuged and both ultrafiltration and cationexchange subfractionation had to be applied
Precipitation
The application of precipitation techniques in oligopeptide analysis has been widely described in the literature [19 38 40 48–56] Biological fluids are often submitted to precipitation methods that allow removal of undesirable compounds from the samples (proteins lipids etc) The selectivity of precipitation strongly depends on the type of precipitating agent used The application of organic solvents such as ethanol methanol and acetonitrile or their mixtures often provides effective protein removal Table 1 shows the precipitation agents that have been applied in oligopeptide analysis The use of cold solvents prevents a thermal stress of the analytes The salting out (ie with ammonium sulfate) also resulted in a selective fractionation and removal of proteins and polypeptides from the complex sample matrices For instance precipitation with acetone was effective in the analysis of peptides (Lcarnosine Lanserine and glutathione) by capillary electrophoresis (CE) [54] This precipitating agent was more suitable than ethanol and sulfosalicylic acid In the case of ethanol higher volumes of the solvent were
Table 1 Examples of precipitation agents in oligopeptide analysis
Analyte
Precipitation agent
Sample matrix
Detection technique
LODLOQ
Ref
Opioid peptides
Perchloric acid
Rat brain
HPLCFL
1 pmolml
[38]
Synthetic peptide
Acetonitrile
Rat serum
LC–MS
02 μgml
[49]
Pentapeptide drug
Acetonitrile
Rabbit and human plasma
2 ngml
[53]
(IRI5 14)
αAmanitin
Acetonitrile
Human plasma
LCMSMSMS
06 ngml
[40]
Mushroom peptide
Acetonitrile
Human plasma
CE
50 ngml
[43]
toxins
Glutathione
Methanol
Human plasma
HPLCFL
02 pmolml
[48]
LCarnosine
Acetone
Human plasma
CE
7 nmolml
[54]
LAnserine
Acetone
Human plasma
CE
10 nmolml
[54]
Glutathione
Acetone
Human plasma
CE
2 nmolml
[54]
Ceftazidime
Trichloroacetic acid
Human plasma
HPLC–DAD
01 μgml
[56]
and iperacillin
Neurotensin [8–13]
Methanolethanol (31)
Human serum
MALDITOFMS
–
[55]
Foodderived peptides
Ethanol
Human plasma
RPHPLC
20 nmolml
[51]
Caseinderived peptides
Calcium bromide and
Cheese
HPLC–MS
–
[36
57]
chloride (CaCl2 and BaCl2)
required which resulted in increased sample dilution and a decrease of detection limits
Another solvent methanol was an effective protein precipitating agent in the determination of glutathione concentration in plasma samples [48] An equal volume of organic solvent (methanol) was added to the plasma and incubated for 30 min to ensure the optimal removal of proteins The samples were then derivatized with monobromobimane Both protein precipitation and derivatization were carried out in a single step The recovery of glutathione was above 94 with a detection limit at 05 ngml
The application of a mixture of solvents can increase the effectiveness of protein precipitation during the monitoring of neurotensin degradation in human and rat serum [55] Precipitation using a mixture of methanol and ethanol (31) has been performed after which the samples were vortexed centrifuged and spotted on the target plate for matrixassisted laser desorptionionization timeofflight mass spectrometry analysis (MALDITOFMS) The limit of quantification was
007 mM
Besides the use of organic solvents oligopeptide deprotenization can be also done by the application of inorganic acids and salt solutions [19 38 56] Among the most often used precipitating agents were trifluoroacetic acid trichloroacetic acid perchloric acid or sulfosalicylic and alginic acid For instance a solution of HClO4 was effective in removing the endogenous biosubstances that interfered with the analysis of opioid peptides from rat brain [38] Nevertheless the use of such a precipitation method was not efficient enough and in addition the solidphase extraction procedure for analyte preconcentration had to be performed
In the analysis of food samples the precipitation methods give the possibility of the selective fractionation of oligopeptides that had been found in the hydrolysates of food proteins [36 52 57] For the determination of cheese peptides the precipitation solutions comprising salts as well as organic solvents have been applied In most analyses the precipitation of oligopeptides under acidic conditions had a better effect compared with inorganic salts albeit mainly in the analysis of hydrophobic analytes [52] Nevertheless trichloroacetic (TCA) and trifluoroacetic acid (TFA) are the classical protein precipitants that have been applied for this purpose It has been observed that the degree of peptide separation depends on the acid concentration For example small oligopeptides were more effectively isolated when a higher concentration of TCA (about 12) was used On the other hand larger polypeptides were more easily isolated when a lower concentration of trichloroacetic acid (about 25) was applied Unfortunately the essential removal of the chlorinated acetic acids prior to further analysis is a rather laborious procedure Therefore the acid solutions are often replaced by the precipitation agents that can be easily removed by evaporation (ie organic solvents)
Inorganic salts such as CaCl2 and NaCl solutions facilitated the precipitation of caseinderived peptides [36] NaCl was less effective as a precipitating agent than CaCl2 Furthermore the addition of BaCl2 at a concentration between 10 and 50 mM at pH about 7 improved the precipitation of highly negatively charged polypeptides (eg phosphonopeptides)
A large number of precipitation procedures utilize mixtures of organic solvents (chloroformmethanol) for the removal of sample interferences in the case of isolation
of oligopeptides from food hydrolysates as has been reviewed by Mc Sweeney and Fox [36] In general chloroform was the main solvent used to remove the interfering lipids from cheese samples Sometimes the combination of methylene chloride methanol and water as well as ethanol containing trichloroacetic acid gave better results However the need for some additional sample cleanup methods such a solidphase extraction and sizeexclusion chromatography to eliminate other interferents was also usually required
Summing up the effectiveness of protein removal by precipitation methods has several limitations Firstly the application of some organic solvents limits sample compatibility with the mobile phases of liquid chromatography which are commonly used for analysis Secondly strong acids or bases are undesirable if the analyzed oligopeptides are sensitive to high or low pH Besides the use of precipitation methods may also increase of chromatographic noise and appearance of the additional ghost signals on chromatograms which can significantly disturb the analysis of oligopeptides Furthermore unprecipitated endogenous compounds remaining in the sample may result in chromatographic column stacking
Nevertheless both homogenization and precipitation are easy and cheap preliminary sample pretreatment techniques because they require the use of inexpensive chemicals usually available in the analytical laboratory In many cases after homogenization andor precipitation the oligopeptide samples were merely centrifuged and analyzed without any additional manipulation However the obtained supernatant usually requires further sample pretreatment techniques that enable mainly the preconcentration of analytes
Advanced sample pretreatment
Very often the supernatant obtained after centrifugation required the use of further sample pretreatment methods for isolation purification and preconcentration of oligopeptides Such a possibility is offered by membranebased systems with ultrafiltration (UF) or dialysis (D) as well as low pressure chromatographic separation techniques (ie sizeexclusion (SEC) or ionexchange chromatography (IEC)) Unfortunately these techniques enable only the isolation and purification of analytes Therefore due to the low concentration level of oligopeptides usually below the detection limit of commonly used analytical instruments the application of an additional enrichment step(s) is required to increase the analyte concentration The simultaneous purification and preconcentration of oligopeptides is possible by application of extraction procedures such as classical liquid–liquid extraction solidphase extraction and supported liquid membrane extraction
Membrane separation techniques
Membrane processes have also been used for the effective fractionation of sample components In the case of analysis of complex sample matrices the membrane separation techniques enable first of all the separation of low molecular compounds from macromolecular substances These methods facilitate the removal of organic and inorganic salts that are so undesirable when MS is used in analysis as a detection system
Among the commonly known membrane techniques ultrafiltration dialysis and microdialysis have found wider application for analysis of oligopeptides [8 20 39 44 58– 64]
Ultrafiltration (UF)
Very often a peptidic fraction obtained after purification by classical methods is still too complex to allow direct analysis by the commonly applied analytical instruments This limitation may be reduced by the application of ultrafiltration techniques In the case of oligopeptide analysis the ultrafiltration methods were mainly useful for isolation and fractionation of analytes of interest from food samples [8 9 20 44 58–61 64] This technique was usually applied to improve the resolution of analytes There are only a few applications in which the ultrafiltration was used as a sample preparation step in analysis of oligopeptides from biological samples [39 65] However because of some limitations it is not used frequently
Generally the membranes used for ultrafiltration processes are made of polysulfone polychlorovinyl and cellulose derivatives Sometimes ceramic membranes can be also utilized For instance a regenerated cellulose membrane was applied for effective fractionation of peptides from a watersoluble extract of Camembert cheese These experiments led to identification of peptides which were responsible for the bitter taste of cheese appearing during its ripening [8 44] More examples of UF analysis that have been used for isolation of oligopeptides from several varieties of cheese were described by McSweeney and Fox [36] and Herraiz [20] In most described cases the membranes with a cutoff range between 05 and 10 kDa was applied Sometimes membranes with low molecular weight cutoffs were useful in isolation of smaller peptides from cheese
Two steps of ultrafiltration processes have been used for fractionation of peptides from tryptic and chymotryptic hydrolysates of whey protein concentrate [64] Firstly the membrane with a cutoff value of 30 kDa was applied to remove proteins and polypeptides from the sample This procedure took about 45 min Afterwards in the second
step the ultrafiltration membranes with 1kDa cutoff were used for fractionation and isolation of small peptides and amino acids from the permeate
In another paper the ultrafiltration membrane in a twoelectrode configuration was applied as an effective system for isolation of strongly charged bioactive peptides from a hydrolysate of α32caseine [61] In these experiments the polysulfone membranes with a cutoff between 10 and 100 kDa were used Only positively charged peptides (besides inorganic salts) reached the permeate (product) No negatively charged or neutral peptides were found in the permeate A 100 purity of product was obtained The total time required for the ultrafiltration process was approximately 4 h Highly nutritive oligopeptides with antimicrobial properties had been studied in this way
In summary the limited application of ultrafiltration for isolation of oligopeptides is mainly caused by the lack of selectivity and problems with membrane clogging Furthermore the ultrafiltration procedures are usually timeconsuming However the fact that the sample is not diluted during the process and does not require organic solvents should be considered as its advantages Therefore an additional enrichment step is usually required to reach a suitable detection limit
Dialysis (DIA)
The other membrane technique that was applied for isolation and purification of oligopeptide samples is dialysis [36 62 66] The commonly used dialysis membranes are mostly made of synthetic polymers or cellulose In analytical chemistry this technique is generally applied in two cases Firstly to isolate various low molecular weight substances from biological fluids and secondly when nonvolatile compounds (inorganic ions) have to be removed from the sample to improve the detection by mass spectrometry
Regarding the oligopeptide analysis only a few methods of dialysis have been developed These were mainly used to fractionate watersoluble peptides from mature cheese [36] There are also some dialysis methods that have been developed for isolation of some oligopeptides from wine and must [62 66] In all cases the dialysis was performed against water Even though the recovery value of analytes was high the time required for those methods (eg 96 h) discouraged the researchers from its further use Therefore this technique was replaced by other more effective sample pretreatment techniques In addition the significant dilution of samples binding of analytes to the membrane material as well as problems with membrane stability and the necessity for salt removal are the main disadvantages of this technique
Microdialysis
In recent years microdialysis methods have attracted substantial interest Briefly this is a technique commonly used in indirect in vitro methods and it enables the determination of concentration of free (ie proteinunbound) analyte Comprehensive reviews providing an overview of the principles and applications of microdialysis in pharmacodynamic studies of local physiological events were published recently [67–71] Here we would like to present some interesting examples considering their compatibility with chromatographic analytical systems
For instance in the case of oligopeptide analysis microdialysis was applied for continuous in vivo monitoring of extracellular glutathione levels in cerebral ischemia [72] The microdialysis catheter consisted of a tubular dialysis membrane with a 20kDa relative atomic mass cutoff Samples were collected every hour or halfhour The obtained microdialysates were offline injected into the highperformance liquid chromatography system The detection limit of glutathione was 01 mgl
The online combination of microdialysis with an HPLC system allowed the determination of glutathione concentrations in the livers of anaesthetized rats [73] The membrane of the microdialysis probe was made of polycarbonate and Ringer’s solution was used as the perfusion fluid that filled the probe’s interior The measured concentration of glutathione in the sample was in the range from 416 to 76 μmoll The microdialysates were collected every 10 min and directly injected into the HPLC Compared with the work of Landolt et al [72] this online method significantly shortened the analysis time
Recently an online system of microdialysis with capillary liquid chromatography and mass spectrometry was developed for the determination of some enkephalins in striatum of anesthetized and freely moving rats [70] The detection limits for the quantification of analytes were approximately 1–2 ngl The reproducibility was below 5 when mass spectrometry in the MSMSMS mode was used In addition a similar system of packed capillary LC– ESITOFMS was also applied for quantification of bradykinins (nonapeptides) in rat muscle tissue [74] In this case the detection limit was 01 ngml
Microdialysis was also applied for the measurement of the in vivo concentrations of glutathione in breast tissue and subcutaneous fat during the menstrual cycle [75] The equilibration of microdialysis catheters with the tissue took 3 h The collected fractions were then analyzed offline by highperformance liquid chromatography with an amperometric detector The detection limit of glutathione was at the picomolar level
To sum up the possibility of online connection with the commonly used analytical instruments is the main advan
tage of microdialysis It enables during surgical operation the in vivo determination of various analytes including oligopeptides Usually no additional sample pretreatment procedures are required However the microdialysis devices used in the methods described in the literature possess rather invasive character and have mainly been applied to the experiments carried out with laboratory animals
Extraction procedures
The low concentration level of analytes usually below the detection limit of commonly used analytical instruments is a significant problem in the case of analysis of oligopeptides from complex sample matrices Therefore the application of supplementary steps for analyte preconcentration is required Unfortunately the preliminary pretreatment stages like homogenization and precipitation as well as membrane separation techniques enable only the isolation and purification of analyte of interest but not its enrichment For that reason in most analyses the application of additional enrichment steps is required to increase the analyte concentration The simultaneous purification and preconcentration of oligopeptides is possible by the application of extraction procedures The classical liquid–liquid extraction and solidphase extraction are most often used
Liquid–liquid extraction
Liquid–liquid extraction (LLE) is the oldest type of extraction technique used commonly in many analytical laboratories Considering oligopeptide analysis liquid– liquid extraction technique has found application mainly in the pretreatment of biological samples (blood plasma and cerebrospinal fluids) [19 35 43] By appropriate selection of the solvent type and pH of an aqueous phase as well as sample ionic strength almost 100 analyte recovery was possible Even though the oligopeptides are polar compounds and extraction into an organic medium is limited the use of an organic solvent or its mixture with some acids usually helps to increase the recovery Selected examples of conditions for liquid–liquid extraction are summarized in Table 2
Liquid–liquid extraction was effectively applied for isolation and preconcentration of synthetic analogs of opioid peptides and substance P from cerebrospinal fluids It helped to remove most of the CSF proteins that interfered with the measurements due to problems with crossreactivity [35] The developed sample pretreatment method highly improved the recovery of neuropeptides at femtomolar concentrations It has been demonstrated that the application of simple liquid–liquid extraction with an organic phase consisting of a mixture of ethyl acetate acetone and petroleum ether enabled the recovery of those analytes with high selectivity Additionally the application of sample acidification with HCl before the liquid–liquid extraction procedure improved the removal of proteins from the sample and significantly increased the enrichment of the analytes For the analysis without HCl about 30–40 higher values of recovery were obtained Compared with the experiments with solidphase SepPak C18 cartridges the application of liquid–liquid extraction increased the enrichment of enkephalin peptides In addition this developed LLE procedure was significantly simpler and more effective than the solidphase extraction (SPE) In this case the analytes were determined at a 2 pgml concentration level
The application of liquid–liquid extraction for isolation and preconcentration of substance Prelated peptides from tissue homogenates and body fluids has been extensively reviewed by Rissler [19] Since those oligopeptides are polar substances their extraction recovery was very low even after performing multiple extractions however the use of various waterimmiscible solvents provided an effective solution A significant increase of analyte enrichment was noticed when an ionpair agent was added (ie TFA) Unfortunately because of the application of mass spectrometry detection in this procedure the removal of inorganic compounds was required
The LLE procedure was also used for the isolation and preconcentration of some cyclic oligopeptide toxins from human body fluids [43] nButanol was successively used for the separation of those compounds from urine samples The total time for liquid–liquid extraction was only 10 min The extraction yields exceeded over 80 In contrast for isolation of these toxins from blood samples the precipitation with acidified acetonitrile followed by centrifugation
Table 2 Selected examples of conditions for liquid–liquid extraction in oligopeptides analysis
Analyte
Sample matrix
Extraction procedure
LODLOQ
Ref
Substance P opioid peptides
CSF
(1) Ethyl acetatepetroleum ether (11)
(2) Acetonepetroleum ether (11)
2 pgml
[35]
Mushroom peptide toxins
Human urine
(3) Ethyl acetate
nButanol
50 ngml
[43]
Substance P
Tissue body fluids
Diethyl ether ethyl acetate methylene chloride hexane
–
[19]
was sufficient The solvent from the extract and the supernatant was removed by rotary evaporation and the samples were analyzed by capillary electrophoresis In both cases the detection limit was about 50 ngml
To conclude despite the simplicity and easy of use of LLE procedures their popularity has decreased in recent years because of numerous disadvantages with this extraction technique eg it is a tedious and timeconsuming procedure that is not easy to automate and emulsion formation is possible during liquid extraction
Solidphase extraction
Solidphase extraction (SPE) is currently the most often used sample pretreatment technique in analytical procedures Compared with liquid–liquid extraction SPE shows lower selectivity but access to many types of sorbents (eg C18 C8 C2 phenyl CN NH2 and ionexchange bonded materials) enables effective and selective isolation and extraction of many classes of compounds The use of reversedphase SPE cartridges makes it possible to moderate and optimize the analyte purification and desalting The addition of small amounts of trifluoroacetic acid to the eluent usually significantly improves the recovery of analytes
Many papers that deal with solidphase extraction applied for oligopeptide analysis have been published recently [7 13 17 40 51 53 62 76–83] In most cases
the complex matrices of analyzed oligopeptides were directly applied to the SPE sorbents and the analytes were then eluted with suitable solutions Sometimes better recovery values were obtained when preliminary precipitating or low pressure chromatographic techniques were used Compared with liquid–liquid extraction methods solidphase extraction provides a more effective sample pretreatment technique for isolation and preconcentration of oligopeptides both from food and biological samples Examples of extraction conditions for solidphase extraction are presented in Table 3
Herreiz and Casal fully reviewed the SPE methods used in the analysis of foodderived peptides [21] Rissler evaluated solidphase extraction procedures applied in the determination of substance Prelated peptides in various types of samples [19] Therefore only some additional examples of SPE application for oligopeptide analysis not included in these reviews will be described below
The octadecyl SPE sorbents were adopted for the quantitative analysis of opioid peptides from human plasma samples [78] The elution was done with a mixture of acetonitrile and trifluoroacetic acid The effluent was then evaporated and the residue was redissolved in a solution of ammonium acetate and analyzed by LC–ESIMSMS The recovery of analytes from plasma was over 80 This method permitted the detection of analytes by utilizing only 025–05 ml of plasma The detection limit of enkephalin peptides in plasma was approximately 1 ngml
Table 3 Selected examples of extraction procedures for solidphase extraction in oligopeptides analysis
Analyte
Sample matrix
Extraction procedure
LODLOQ (ngml)
Ref
Synthetic drug
Human plasma and urine
Sorbent DiolSPE
10
[76]
Cetrorelix
Human plasma and urine
Elution 025 mM NaOH and 1 CF3CO2H in CH3OH
Sorbent C8
2
[13]
Opioid peptides
Human plasma
Elution CH3OH H2O CF3CO2H (901001)
Sorbent C18
1
[79
81]
IRI5 14 (drug)
Rabbit and human plasma
Elution 80 CH3CN01 CF3CO2H
Sorbent C18
2
[53]
Antagonist G
Human plasma
Elution (1) 01 TFA (2) 3 CH3CN in 01 CF3CO2H
Sorbent C2
20
[17]
αAmanitin
Human serum kidney
Elution CH3OH 1 M CH3COONH4
Sorbent C18benzenesulfonic acid
02
[40]
Angiotensin II
and liver
Human plasma
Elution CH3OH
Sorbent CBA
20
[83]
Bioactive
Marine organism
Elution 100 mM CH3COONH4 in 90 CH3OH
Sorbent Amberlite XAD
–
[7]
oligopeptides
Enkephalins
Cerebrospinal fluids
Elution methanolwater
Sorbent C18
100
[77]
Enkephalins
Human plasma
Elution 16 mM ammonium acetate buffer pH 58
Sorbent C18
025
[78]
Cyclic peptides
Rat plasma
Elution 80 acetonitrile01 CF3CO2H
Sorbent C18
6
[80]
Elution 70 acetonitrile30 water
In clinical analysis of peptide drugs highly specific and sensitive methods must be used since limited bioavailability of the drugs often results in low drug levels in plasma Sensitive quantification of the pentapeptide drug IRI5 14 (AcArgProAspProPheNH2) in plasma by using highperformance liquid chromatography coupled with ESIMS has been described in the literature [53] Prior to the analysis the plasma samples were prepared using protein precipitation followed by solidphase extraction The precipitation was used for plasma protein removal whereas SPE C18 sorbent enabled the elimination of the excess of salts and interferents For preconcentration and elution of IRI5 14 a mixture of acetonitrile and 1TFA in water was the most effective The analyte recovery from plasma samples was over 70 The lowest limit of quantification was 2 ngml
Another interesting example of using extraction is the isolation of αamanitin from liver and kidney samples which was possible by the application of homogenization with a mixture of 01 M phosphate buffer (30) and acetonitrile (70) without any analyte loss [40] The obtained homogenate was then centrifuged and the supernatant was shaken with methylene chloride to remove the acetonitrile from the sample Finally the aqueous phase was cleaned up using the solidphase extraction procedure and analyzed by HPLCMS
A microbore analytical column with a C2 sorbent was used for removal of interfering components from blood plasma samples in the case of determination of the anticancer peptide drug (antagonist G) and its three major metabolites [17] The elution of analytes from the SPE column was done with methanol and 1 M ammonium acetate mixture (9010 vv) The limit of quantitation was 20 ngml for antagonist G and 100 ngml for its metabolites
Mixedmode C18benzenesulfonic acid solidphase extraction cartridges were used in a sample pretreatment procedure for the determination of αamanitin in serum and tissues samples (kidney and liver) [40] The SPE procedure was preceded by precipitation in order to remove the interfering substances from the samples The analyte was eluted with methanol evaporated to dryness redissolved in water and injected into an HPLC system Recovery of the analyzed compound was over 98 The detection limit of the developed method was 020 ngml
The solidphase extraction method based on a weak cationexchange mechanism with the use of silica sorbents modified with carboxylic acid (CBA) has been applied for the purification of angiotensin II and gonadoliberins in human plasma [83] Sample impurities were washed off with a mixture of ammonium acetate in water and ammonium hydroxide in acetonitrile Elution took place with a solution of ammonium acetate in methanol Volatile
solvents were irreplaceable for effective removal of undesirable salts from the plasma samples The detection limit for studied analytes was 50 ngml In addition in another work it has been proven that the enrichment factors of analyzed enkephalins from plasma were about 20 higher when C18bonded material was applied compared with a strong cationexchange column (SCX) [80] Unfortunately the high level of salt content in the case of analysis with SCX was unsuitable for the chromatographic instrument used in such experiments
Solidphase extraction methods were mainly used for peptide isolation and fractionation in the case of food sample analysis rather than body fluid samples Usually the high concentration of oligopeptides in protein hydrolysates did not required the use of preconcentration steps Extensive review of the application of SPE methods in food sample analysis has been presented in a paper by Herraiz and Casal [21] and therefore it will not be described in detail Nevertheless it may be concluded that hydrophobic oligopeptides in food are usually extracted with the use of octadecyl (C18) sorbents CN sorbents were adequate for very hydrophilic compounds In the case of nonpolar and hydrophobic analytes the application of a wide spectrum of SPE cartridges such as PH CH C8 and C2 may be useful More details about appropriate selection of sorbents for solidphase extraction of peptides in the case of food analysis may be found in the aforementioned paper
To summarize the solidphase extraction technique is one of the most often used methods in sample pretreatment procedures Unfortunately the use of substantial amounts of organic solvents and the necessity of carrying out many laborious operations are the main disadvantages of this method Furthermore this method is characterized by low selectivity Generally the separation of analytes is based on the hydrophobicity Therefore development of new faster and more selective methods for sample cleanup and enrichment of various analytes from complex matrices is still needed
Membrane extraction techniques
Supported liquid membrane extraction (SLM) may provide an alternative to SPE sample pretreatment methods SLM combines the advantages of classical membrane processes and liquid–liquid extraction offers the possibility to perform separation and preconcentration in one step and uses small volumes of organic solvents (only for making up the liquid membrane)
One method has recently been reported for the SLMbased analysis of short peptides from plasma samples [84] In this procedure SLM was connected online to RPHPLC with UV detection and it enabled the extraction and preconcentration of small model peptides containing from
two to six amino acids including chemotactic (MetLeuPhe) and immunostimulating (ValGluProIleProTyr) peptides in a fully automated system The transport of permanently charged peptides across hydrophobic organic membrane was facilitated by an anionic carrier (Aliquat 336 a quaternary ammonium salt) in the membrane A gradient of chloride anions was a driving force for the process The minimum quantifiable concentration of investigated analytes was in range 90–130 ngml based on 05 ml of plasma The problem of protein interaction with the liquid membrane surface was overcome by using a short ultrafiltration procedure
Low pressure chromatographic methods
A large number of currently available analytical methods for quantification of oligopeptides in complex sample matrices utilize offline sample pretreatment procedures However the online combination of two or more purification techniques is a powerful tool for the determination of peptides Such possibilities may be achieved by the application of low pressure chromatographic techniques including sizeexclusion chromatography (SEC) and ionexchange chromatography (IEC) The use of a small reversedphase cleanup column prior to the application of a mass spectrometry system has also gained interest in recent years The online coupling of at least two techniques in a fully automated system significantly shortens the analysis time and limits possible sample contamination Moreover such a multidimensional system enables the required resolution andor selectivity to be achieved in one step
In the case of sizeexclusion chromatography (SEC) the simultaneous removal of salts and interferents was possible [27 59 85–89] Moreover SEC allowed peptide fractionation that is required in the case of analysis of protein hydrolysates [45 51 62 90–92] Various types of resin like silica crosslinked dextran (Sephadex) and copolymers of divinylbenzene and its sulfate analogues (with various pore size) were used as column stationary phases Depending on the type of resin used oligopeptides were eluted from the column with water organic acids (ie formic and acetic acid) as well as solutions of ammonium acetate or bicarbonate Sometimes the use of a mobile phase that contained alcoholic solutions helped to reduce the potential hydrophobic interaction of the analyte and the silica
Ionexchange chromatography (IEC) permits the fractionation of complex sample components according to the differences in charge This technique was particularly well suited for the isolation of peptides since most peptides possess net charge at side chains or the N or Cterminus The stationary phases commonly used in IEC are resin or
gel matrices that consist of agarose or cellulose beads with covalently bonded charged functional groups respectively Elution of the analyte was usually achieved by increasing the ionic strength of the mobile phase in order to break up the ionic interaction or by changing the pH of analyzed samples There are several reports of selective elution processes in peptide purification [39 93 94] However similar to the case of the application of sizeexclusion chromatography most of them concern the area of food sample analysis [36]
An online system of sizeexclusion chromatography (SEC) and reversedphase liquid chromatography (RPHPLC) was developed for the quantification of structurally related enkephalins in cerebrospinal fluid samples [88] The SEC column was packed with hydrophilically modified silica for the first dimension and C18 for the second A water solution containing glacial acetic acid and ammonium acetate was used as the mobile phase in the first dimension The analytes were eluted under isocratic conditions using acetonitrile as an organic modifier of the mobile phase in the case of the second dimension The limit of detection (LOD) was 5 ngml Unfortunately the analysis of CSE spiked with analytes caused the occurrence of serious interferences between the signals of analytes The increase of the injection volume did not improve the limit of quantification The same SEC conditions were applied for the determination of enkephalin peptides in plasma and cerebrospinal fluid samples [85] After the separation of enkephalins from the sample proteins in the SEC dimension the fraction of analytes was concentrated on the top of the RPHPLC column and finally injected into a capillary electrophoresis system The developed procedure was fully automated Furthermore compared with the earlier described system the quantitation limits of the studied analytes were slightly lower
In the case of a determination of substance P in the rat ventral tagemental area a complex sample pretreatment procedure that involved homogenization extraction and an online system of sizeexclusion chromatography and ionexchange chromatography has been also described [89] The SEC column was packed with Superdex (dextran chains covalently linked to an agarose matrix) Ammonium bicarbonate was used for analyte elution The active fractions were pooled for further separation on a strong ionexchange column (Resource Q) based on rigid beads made of polystyrenedivinyl benzene In this dimension the sample was eluted with sodium phosphate buffer (pH 74) The obtained final fraction of analyte was assayed for SP endopeptidase activity
Sizeexclusion chromatography in an online system with solidphase extraction and capillary electrophoresis has been applied to remove the interfering compounds from a sample of cerebrospinal fluid [77] In these experiments the
application of a SEC column was necessary to eliminate the undesirable proteins present in the samples of cerebrospinal fluids For SEC experiments a highefficiency silicabased gel filtration column was selected For the effective elution of analytes ammonium acetate buffer with pH 58 was utilized The detection limits of the analyzed enkephalins were about 100 ngml
Sephadex G25 column based on crosslinked dextran was used for separation and determination of foodderived oligopeptides in plasma samples [51] In this research the solution of the peptide fraction obtained from the SEC column was then derivatized with phenyl isiothiocyanate to facilitate their specific detection Unfortunately the sizeexclusion chromatography procedure permitted only the removal of plasma proteins from the analyzed samples To improve the resolution of the analytes the solidphase extraction procedure for analyte preconcentration had to be applied The recovery of foodderived peptides was over 70 after this stage The studied analytes were determined at 20 pgl concentration
In the case of food sample analysis the content of peptides compared with biological samples is much higher and an enrichment step is usually not required Therefore the application of only one chromatographic technique for fractionation of sample compounds is sufficient For instance such a method was applied for the purification of antioxidant peptide fractions from mackerel hydrolysates where the use of only sizeexclusion chromatography was required [45] The hydrolysate was fractionated on a Sephadex25 column and eluted with deionized water Three major fractions were obtained The estimated size of these components was 1400 900 and 200 Da The 1 400Da fraction possessed the strongest antioxidant activity
Sephadex25 resin has also been utilized for sizeexclusion chromatography experiments in the case of structural analysis of antioxidant peptides from soybean protein (βconglycinin) A 50 mM acetic acid solution was used for elution of analytes from the SEC column Before the application of mass spectrometry detection the analytes were additionally purified on a semipreparative HPLC column
The Sephadex resin has also found application for the separation of peptides from wine samples This technique enabled simultaneous removal of both small and higher molecular weight interfering components from the analyzed samples Water solutions of acetic acid sodium chloride and aqueous organic solvents mixtures were very useful for peptide elution Furthermore these developed procedures were valid for the analysis of peptides with low molecular weight (<1000 Da)
The use of silicabased strong cationexchange sorbents for purification of some synthetic and proteolytically
derived peptide fragments was described in the literature [93] The sulfoethyl aspartamide resin was utilized as a column stationary phase A gradient elution of acetonitrile and methanol with 85 phosphoric acid was used for IEC The desirable peptide fraction was usually collected at 05to 10min intervals over the entire gradient Nevertheless the application of semipermeable RPHPLC for desalting was necessary in order to perform the mass spectrometry analysis
The work of Sommerer at al [59] is a representative example of the application of a sequential chromatography system to the isolation and purification of cheese peptides In this case the ultrafiltered watersoluble extract was successfully submitted to sizeexclusion chromatography anionexchange chromatography and a mass spectrometry followed by semipreparative reversedphase highperformance liquid chromatography The peptide analytes were eluted with water The IEC fractions were collected manually and after drying under vacuum the samples were dissolved in water and injected onto a semipreparative RP18HPLC column to remove salts from the sample before the mass spectrometry detection Using this method the sequences of 28 short peptides have been obtained
To sum up the application of low pressure liquid chromatography techniques permits mainly fractionation of analytes and removal of sample interferences such as proteins detergents or salts from complex sample matrices Therefore the use of additional sample pretreatment procedures for analyte preconcentration in the case of oligopeptide analysis is required However the disadvantages like significant sample dilution and loss of polar peptides due to their adsorption on the polar crosslinked dextran surface limit the applicability of such techniques
Conclusion
Currently much scientific research focuses on the identification quantification and purification of natural and synthetic peptides The occurrence of peptides in a wide range of samples highlights the need for their analysis in samples of different origin The complexity of the studied material usually requires the application of sample pretreatment techniques for isolation purification and preconcentration of the analyte Combining two or more sample pretreatment procedures usually improves the overall resolution and sensitivity
Examples of sample pretreatment techniques that enable effective and selective isolation andor preconcentration of oligopeptides from complex sample matrices have been reviewed In general the samples of tissue food and biological fluids are first subjected to a preliminary sample cleanup eg homogenization and precipitation to remove
the endogenous interferents (ie proteins lipids and in (organic) salts) from the sample The membranebased techniques and low pressure chromatographic methods for separation and purification of the analytes are then applied The peptide concentration in samples of biological fluids is often at the femtomolar level Therefore the application of extraction procedures for analyte preconcentration is usually required Currently online sample preparation methods and devices tend to be the focus point of research and commercialization
Acknowledgements The authors would like to thank Dr Pawe Dygiel (Institute of Chemistry Opole University Poland) for helpful discussions and suggestions
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Keywords Body fluids Oligopeptides Sample preparation
Introduction
Although the discovery of oligopeptides at the turn of nineteenth and twentieth centuries as products of the protein hydrolysis initially did not draw too much attention the significant role of this group of compounds in the functioning of all living organisms has since become well known Nowadays it is generally accepted that the existence of oligopeptides in the human body is indispensable for the proper functioning of numerous internal biological systems [1 2] Oligopeptides include hormones neurotransmitters metabolic regulators antibiotics antioxidants antalgic drugs or even toxins [2–7] Moreover
A Poliwoda (*) P P Wieczorek Institute of Chemistry Opole University Pl Kopernika 11
45–040 Opole Poland
these compounds play an important role in preservation of the rheological properties of food [8 9] Oligopeptides as a final product of food hydrolysis (mostly milk cheese and wine) are known to act as very good antioxidants anticoagulants and blood pressure reducers [10–12] The importance of synthetic oligopeptides has also grown in recent years and these molecules have found wide application in medicine including the therapy of some incurable diseases [13–17]
The widespread occurrence and biological activity of oligopeptides brought about the necessity to determine their presence and concentration in various types of samples Usually the analysis has to be done on very complex matrices such as body fluids (blood plasma serum cerebrospinal fluid or urine) as well as protein hydrolysates of food These samples contain a large variety of different endogenous compounds so most analytical systems are unable to handle them simultaneously Almost without exception oligopeptides are present in such samples at low concentrations together with interfering endogenous large substances (proteins and lipids) that make them difficult to isolate and determine with good accuracy Furthermore the interfering substances can often be confused with the peptides For that reason even with the access of advanced separation and detection techniques it is rather difficult to analyze oligopeptide samples without an adequate sample preparation step The application of specific procedures for isolation purification and preconcentration of analytes is commonly required Due to the complexity of matrices it is often not sufficient to use one sample preparation technique so several methods have to be applied to meet the requirements of proper analysis
Thus far not many papers have been published dealing with techniques for sample preparation in analysis of oligopeptides from complex sample matrices [7 18 19]
There are only a few reports describing sample cleanup procedures applied for the analysis of foodderived peptides [20–22] and these have mainly dealt with specific analytical techniques (eg capillary electrophoresis and HPLC) or a specific group of peptides generally with long amino acid chains [23–26] Only a few communications have described details about applications of only one specific sample pretreatment technique in peptide analysis [21 27 28] The data on sample preparation techniques used for the analysis of small peptides (oligopeptides) are very limited This review is therefore focused on commonly used sample cleanup methods that have been applied for isolation and preconcentration of oligopeptides from various complex matrices In each case examples of applications will be considered and discussed taking into account the selectivity enrichment method automation cleanup and environmental aspects of the developed methods
Analysis on crude samples
The perfect situation for each analyst is to have a method that enables a direct analysis without the need for any additional steps Unfortunately such a situation happens very rarely and is limited to only a few analytical detection techniques like fluorescence detection and immunoassays (RIA ELISA) For example HPLC analysis with fluorescence detection was applied to the determination of a synthetic octapeptide used as an antiviral drug in rabbit plasma samples [16] This compound contains the naturally fluorescent tyrosine residue so no derivatization was required The analyzed plasma samples were only diluted with water before direct injection The sensitivity of this method was about 5 μgl or even lower However the analysis involving naturally present fluorescent components is not very common in the case of oligopeptides Usually for the fluorescence detection technique the use of a fluorescent derivatization agent is essential [29–31]
A few methods that utilized a variety of fluorescent derivatization schemes for the detection of oligopeptides by capillary electrophoresis have been described [30 31] As a simple example the method for the determination of glutathione (GSH) in must and wine can be mentioned [30] In these experiments besides the use of a 20minlong derivatization procedure the must and wine samples were only centrifuged prior to analysis The detection limit was 20 μgl However the method concerned only the analysis of samples of white wine which contains smaller amounts of various interferents that may give a false response and decrease the recovery of analytes
Immunological methods allow detection of peptides at very low concentrations and with high specificity for a given analyte Unfortunately the use of immunological
detection in the case of peptide analysis is limited only to compounds against which specific antibodies have been generated For example the direct injection of complex sample matrices (plasma) without any manipulation was used in the analysis of larger polypeptides (28–32 amino acids) against which specific antibodies have been produced [32–34] However the specificity of immunological assays may be questionable especially if they are not combined with HPLC For instance the presence of proteins and salts in biological samples often results in RIA assay inaccuracy In addition problems with crossreactivity of a specific antibody with compounds that are structurally similar to the analytes forces the use of some sample cleanup steps Such a problem was observed during the determination of some opioid peptides and substance P in human cerebrospinal fluid (CFS) [35] The greater amount of albumin in the CSF sample interfered in the assay and caused an overestimation of neuropeptide levels when the RIA was done without a prior separation step Therefore it was impossible to determine these oligopeptides directly To overcome this problem a liquid–liquid extraction method was used with success
To conclude in the case of oligopeptide analysis there are only few examples of direct injection procedures of complex sample matrices into analytical instruments Usually the complexity of various interfering compounds makes oligopeptide analysis impossible Hence the use of a suitable sample pretreatment procedure is required For instance the presence of some colorants preservatives and antioxidants in food samples (like in red wine samples) limited the effectiveness of direct fluorescence analysis Furthermore the concomitant derivatization of plasma interferents in oligopeptide analysis caused decreased sensitivity Thus in order to avoid these limitations additional methods for removal of endogenous compounds and oligopeptide preconcentration are almost always necessary Taking into account that usually complex samples are analyzed (eg blood urine or food) a direct analysis could be done in only a few cases when the content of analytes of interest in the sample was high
Preliminary sample cleanup
Several cleanup methods are currently available when samples containing oligopeptides need to be characterized and these furthermore depend on the type of sample to be analyzed (liquid or solid) however the need to use many operating steps during the sample pretreatment procedure increases the probability of sample contamination Therefore the possibility of applying simple preliminary pretreatment methods like homogenization precipitation and filtration for preseparation of the analyte of interest
from a complex matrix may be a more desirable approach The application of a simple preliminary pretreatment step like homogenization andor precipitation can sometimes be sufficient
Homogenization
Homogenization of tissue samples such as a brain liver kidney or food (meat and cheese) is the first step of sample pretreatment commonly used before protein precipitation enzymatic digestions or extraction techniques Sometimes some of the large proteins are removed by the application of filtration after sample homogenization Several methods for homogenization of samples containing oligopeptides have been described in the literature [9 19 36–45]
Generally the biological tissues containing oligopeptides are homogenized in solution of saline or buffers [19 38 39 41] It has been noticed that the stability of analytes depends on the pH of applied buffer as well as type and concentration of organic acid For example the superiority of trifluoroacetic acid over trichloroacetic acid has been proven [46] When homogenization and sample storage were performed at 4 °C structural changes of the peptides were not observed [47] The use of buffers and a mixture of organic solvents helps in isolation of very hydrophobic peptides [40] Complete homogenization of tissues was usually achieved in 10–30 s which was an additional advantage of this technique In all cases the application of homogenization helped to prepare a uniform mixture and disperse minute fragments of tissue Sometimes homogenization and centrifugation were sufficient for effective sample cleanup as was accomplished in an investigation of angiotensin II formation in the human heart interstitium [41] The obtained heart homogenates (NaCl at pH 74 and a temperature of 4 °C for 1 min) were centrifuged and the supernatant was then evaporated to dryness dissolved in 01 trifluoroacetic acid solution and injected directly into an HPLC instrument without any problems
Regarding the analysis of oligopeptides from food samples homogenization methods were mostly applied on cheese samples [36 42 44] In general water and aqueous buffers were the most commonly used for extraction of oligopeptides from cheese No influence of homogenization temperature on the level of extractable peptides has been observed The recommended ratio of water to cheese was 21 which afforded high values of analytes recovery even over 90 when the homogenization process was performed in two repetitions In many cases the sample obtained after homogenization was merely filtered and directly injected
Homogenization is therefore rather a supplementary and necessary preliminary step used for isolation of oligopeptides from solid and semisoft samples For illustration
homogenization in water for only 15 s was applied for quantitative determination of the two antihypertensive tripeptides ValProPro and IleProPro from various types of cheese [42] Owing to the high content of the two bioactive peptides in the studied samples no further cleanup and preconcentration stage was required After the homogenization the sample was centrifuged and the filtered supernatant was directly analyzed by HPLCMS Homogenization was therefore a fast and sometimes very efficient method of sample pretreatment for oligopeptides analysis However its application is limited and depends mainly on the oligopeptide content which has to be high to enable determination Usually after homogenization the application of some other sample pretreatment techniques is required in order to obtain a proper sample cleanup andor oligopeptide preconcentration
The preliminary process of homogenization with distilled water and hydrolysis was not enough to determine the small antioxidant oligopeptides from a protein hydrolysate of mackerel [45] Therefore the protein hydrolysate was additionally homogenized with cold 10 trichloroacetic acid a typical precipitation agent However the complexity of the sample required the use of a liquid–liquid extraction step to increase the analyte recovery as will be discussed later A similar situation occurred in the case of isolation of some regulatory peptides from porcine brain [39] In this method after homogenization (with icecold acetic acid ascorbate and ethylenediaminetetraacetic acid) the samples were centrifuged and both ultrafiltration and cationexchange subfractionation had to be applied
Precipitation
The application of precipitation techniques in oligopeptide analysis has been widely described in the literature [19 38 40 48–56] Biological fluids are often submitted to precipitation methods that allow removal of undesirable compounds from the samples (proteins lipids etc) The selectivity of precipitation strongly depends on the type of precipitating agent used The application of organic solvents such as ethanol methanol and acetonitrile or their mixtures often provides effective protein removal Table 1 shows the precipitation agents that have been applied in oligopeptide analysis The use of cold solvents prevents a thermal stress of the analytes The salting out (ie with ammonium sulfate) also resulted in a selective fractionation and removal of proteins and polypeptides from the complex sample matrices For instance precipitation with acetone was effective in the analysis of peptides (Lcarnosine Lanserine and glutathione) by capillary electrophoresis (CE) [54] This precipitating agent was more suitable than ethanol and sulfosalicylic acid In the case of ethanol higher volumes of the solvent were
Table 1 Examples of precipitation agents in oligopeptide analysis
Analyte
Precipitation agent
Sample matrix
Detection technique
LODLOQ
Ref
Opioid peptides
Perchloric acid
Rat brain
HPLCFL
1 pmolml
[38]
Synthetic peptide
Acetonitrile
Rat serum
LC–MS
02 μgml
[49]
Pentapeptide drug
Acetonitrile
Rabbit and human plasma
2 ngml
[53]
(IRI5 14)
αAmanitin
Acetonitrile
Human plasma
LCMSMSMS
06 ngml
[40]
Mushroom peptide
Acetonitrile
Human plasma
CE
50 ngml
[43]
toxins
Glutathione
Methanol
Human plasma
HPLCFL
02 pmolml
[48]
LCarnosine
Acetone
Human plasma
CE
7 nmolml
[54]
LAnserine
Acetone
Human plasma
CE
10 nmolml
[54]
Glutathione
Acetone
Human plasma
CE
2 nmolml
[54]
Ceftazidime
Trichloroacetic acid
Human plasma
HPLC–DAD
01 μgml
[56]
and iperacillin
Neurotensin [8–13]
Methanolethanol (31)
Human serum
MALDITOFMS
–
[55]
Foodderived peptides
Ethanol
Human plasma
RPHPLC
20 nmolml
[51]
Caseinderived peptides
Calcium bromide and
Cheese
HPLC–MS
–
[36
57]
chloride (CaCl2 and BaCl2)
required which resulted in increased sample dilution and a decrease of detection limits
Another solvent methanol was an effective protein precipitating agent in the determination of glutathione concentration in plasma samples [48] An equal volume of organic solvent (methanol) was added to the plasma and incubated for 30 min to ensure the optimal removal of proteins The samples were then derivatized with monobromobimane Both protein precipitation and derivatization were carried out in a single step The recovery of glutathione was above 94 with a detection limit at 05 ngml
The application of a mixture of solvents can increase the effectiveness of protein precipitation during the monitoring of neurotensin degradation in human and rat serum [55] Precipitation using a mixture of methanol and ethanol (31) has been performed after which the samples were vortexed centrifuged and spotted on the target plate for matrixassisted laser desorptionionization timeofflight mass spectrometry analysis (MALDITOFMS) The limit of quantification was
007 mM
Besides the use of organic solvents oligopeptide deprotenization can be also done by the application of inorganic acids and salt solutions [19 38 56] Among the most often used precipitating agents were trifluoroacetic acid trichloroacetic acid perchloric acid or sulfosalicylic and alginic acid For instance a solution of HClO4 was effective in removing the endogenous biosubstances that interfered with the analysis of opioid peptides from rat brain [38] Nevertheless the use of such a precipitation method was not efficient enough and in addition the solidphase extraction procedure for analyte preconcentration had to be performed
In the analysis of food samples the precipitation methods give the possibility of the selective fractionation of oligopeptides that had been found in the hydrolysates of food proteins [36 52 57] For the determination of cheese peptides the precipitation solutions comprising salts as well as organic solvents have been applied In most analyses the precipitation of oligopeptides under acidic conditions had a better effect compared with inorganic salts albeit mainly in the analysis of hydrophobic analytes [52] Nevertheless trichloroacetic (TCA) and trifluoroacetic acid (TFA) are the classical protein precipitants that have been applied for this purpose It has been observed that the degree of peptide separation depends on the acid concentration For example small oligopeptides were more effectively isolated when a higher concentration of TCA (about 12) was used On the other hand larger polypeptides were more easily isolated when a lower concentration of trichloroacetic acid (about 25) was applied Unfortunately the essential removal of the chlorinated acetic acids prior to further analysis is a rather laborious procedure Therefore the acid solutions are often replaced by the precipitation agents that can be easily removed by evaporation (ie organic solvents)
Inorganic salts such as CaCl2 and NaCl solutions facilitated the precipitation of caseinderived peptides [36] NaCl was less effective as a precipitating agent than CaCl2 Furthermore the addition of BaCl2 at a concentration between 10 and 50 mM at pH about 7 improved the precipitation of highly negatively charged polypeptides (eg phosphonopeptides)
A large number of precipitation procedures utilize mixtures of organic solvents (chloroformmethanol) for the removal of sample interferences in the case of isolation
of oligopeptides from food hydrolysates as has been reviewed by Mc Sweeney and Fox [36] In general chloroform was the main solvent used to remove the interfering lipids from cheese samples Sometimes the combination of methylene chloride methanol and water as well as ethanol containing trichloroacetic acid gave better results However the need for some additional sample cleanup methods such a solidphase extraction and sizeexclusion chromatography to eliminate other interferents was also usually required
Summing up the effectiveness of protein removal by precipitation methods has several limitations Firstly the application of some organic solvents limits sample compatibility with the mobile phases of liquid chromatography which are commonly used for analysis Secondly strong acids or bases are undesirable if the analyzed oligopeptides are sensitive to high or low pH Besides the use of precipitation methods may also increase of chromatographic noise and appearance of the additional ghost signals on chromatograms which can significantly disturb the analysis of oligopeptides Furthermore unprecipitated endogenous compounds remaining in the sample may result in chromatographic column stacking
Nevertheless both homogenization and precipitation are easy and cheap preliminary sample pretreatment techniques because they require the use of inexpensive chemicals usually available in the analytical laboratory In many cases after homogenization andor precipitation the oligopeptide samples were merely centrifuged and analyzed without any additional manipulation However the obtained supernatant usually requires further sample pretreatment techniques that enable mainly the preconcentration of analytes
Advanced sample pretreatment
Very often the supernatant obtained after centrifugation required the use of further sample pretreatment methods for isolation purification and preconcentration of oligopeptides Such a possibility is offered by membranebased systems with ultrafiltration (UF) or dialysis (D) as well as low pressure chromatographic separation techniques (ie sizeexclusion (SEC) or ionexchange chromatography (IEC)) Unfortunately these techniques enable only the isolation and purification of analytes Therefore due to the low concentration level of oligopeptides usually below the detection limit of commonly used analytical instruments the application of an additional enrichment step(s) is required to increase the analyte concentration The simultaneous purification and preconcentration of oligopeptides is possible by application of extraction procedures such as classical liquid–liquid extraction solidphase extraction and supported liquid membrane extraction
Membrane separation techniques
Membrane processes have also been used for the effective fractionation of sample components In the case of analysis of complex sample matrices the membrane separation techniques enable first of all the separation of low molecular compounds from macromolecular substances These methods facilitate the removal of organic and inorganic salts that are so undesirable when MS is used in analysis as a detection system
Among the commonly known membrane techniques ultrafiltration dialysis and microdialysis have found wider application for analysis of oligopeptides [8 20 39 44 58– 64]
Ultrafiltration (UF)
Very often a peptidic fraction obtained after purification by classical methods is still too complex to allow direct analysis by the commonly applied analytical instruments This limitation may be reduced by the application of ultrafiltration techniques In the case of oligopeptide analysis the ultrafiltration methods were mainly useful for isolation and fractionation of analytes of interest from food samples [8 9 20 44 58–61 64] This technique was usually applied to improve the resolution of analytes There are only a few applications in which the ultrafiltration was used as a sample preparation step in analysis of oligopeptides from biological samples [39 65] However because of some limitations it is not used frequently
Generally the membranes used for ultrafiltration processes are made of polysulfone polychlorovinyl and cellulose derivatives Sometimes ceramic membranes can be also utilized For instance a regenerated cellulose membrane was applied for effective fractionation of peptides from a watersoluble extract of Camembert cheese These experiments led to identification of peptides which were responsible for the bitter taste of cheese appearing during its ripening [8 44] More examples of UF analysis that have been used for isolation of oligopeptides from several varieties of cheese were described by McSweeney and Fox [36] and Herraiz [20] In most described cases the membranes with a cutoff range between 05 and 10 kDa was applied Sometimes membranes with low molecular weight cutoffs were useful in isolation of smaller peptides from cheese
Two steps of ultrafiltration processes have been used for fractionation of peptides from tryptic and chymotryptic hydrolysates of whey protein concentrate [64] Firstly the membrane with a cutoff value of 30 kDa was applied to remove proteins and polypeptides from the sample This procedure took about 45 min Afterwards in the second
step the ultrafiltration membranes with 1kDa cutoff were used for fractionation and isolation of small peptides and amino acids from the permeate
In another paper the ultrafiltration membrane in a twoelectrode configuration was applied as an effective system for isolation of strongly charged bioactive peptides from a hydrolysate of α32caseine [61] In these experiments the polysulfone membranes with a cutoff between 10 and 100 kDa were used Only positively charged peptides (besides inorganic salts) reached the permeate (product) No negatively charged or neutral peptides were found in the permeate A 100 purity of product was obtained The total time required for the ultrafiltration process was approximately 4 h Highly nutritive oligopeptides with antimicrobial properties had been studied in this way
In summary the limited application of ultrafiltration for isolation of oligopeptides is mainly caused by the lack of selectivity and problems with membrane clogging Furthermore the ultrafiltration procedures are usually timeconsuming However the fact that the sample is not diluted during the process and does not require organic solvents should be considered as its advantages Therefore an additional enrichment step is usually required to reach a suitable detection limit
Dialysis (DIA)
The other membrane technique that was applied for isolation and purification of oligopeptide samples is dialysis [36 62 66] The commonly used dialysis membranes are mostly made of synthetic polymers or cellulose In analytical chemistry this technique is generally applied in two cases Firstly to isolate various low molecular weight substances from biological fluids and secondly when nonvolatile compounds (inorganic ions) have to be removed from the sample to improve the detection by mass spectrometry
Regarding the oligopeptide analysis only a few methods of dialysis have been developed These were mainly used to fractionate watersoluble peptides from mature cheese [36] There are also some dialysis methods that have been developed for isolation of some oligopeptides from wine and must [62 66] In all cases the dialysis was performed against water Even though the recovery value of analytes was high the time required for those methods (eg 96 h) discouraged the researchers from its further use Therefore this technique was replaced by other more effective sample pretreatment techniques In addition the significant dilution of samples binding of analytes to the membrane material as well as problems with membrane stability and the necessity for salt removal are the main disadvantages of this technique
Microdialysis
In recent years microdialysis methods have attracted substantial interest Briefly this is a technique commonly used in indirect in vitro methods and it enables the determination of concentration of free (ie proteinunbound) analyte Comprehensive reviews providing an overview of the principles and applications of microdialysis in pharmacodynamic studies of local physiological events were published recently [67–71] Here we would like to present some interesting examples considering their compatibility with chromatographic analytical systems
For instance in the case of oligopeptide analysis microdialysis was applied for continuous in vivo monitoring of extracellular glutathione levels in cerebral ischemia [72] The microdialysis catheter consisted of a tubular dialysis membrane with a 20kDa relative atomic mass cutoff Samples were collected every hour or halfhour The obtained microdialysates were offline injected into the highperformance liquid chromatography system The detection limit of glutathione was 01 mgl
The online combination of microdialysis with an HPLC system allowed the determination of glutathione concentrations in the livers of anaesthetized rats [73] The membrane of the microdialysis probe was made of polycarbonate and Ringer’s solution was used as the perfusion fluid that filled the probe’s interior The measured concentration of glutathione in the sample was in the range from 416 to 76 μmoll The microdialysates were collected every 10 min and directly injected into the HPLC Compared with the work of Landolt et al [72] this online method significantly shortened the analysis time
Recently an online system of microdialysis with capillary liquid chromatography and mass spectrometry was developed for the determination of some enkephalins in striatum of anesthetized and freely moving rats [70] The detection limits for the quantification of analytes were approximately 1–2 ngl The reproducibility was below 5 when mass spectrometry in the MSMSMS mode was used In addition a similar system of packed capillary LC– ESITOFMS was also applied for quantification of bradykinins (nonapeptides) in rat muscle tissue [74] In this case the detection limit was 01 ngml
Microdialysis was also applied for the measurement of the in vivo concentrations of glutathione in breast tissue and subcutaneous fat during the menstrual cycle [75] The equilibration of microdialysis catheters with the tissue took 3 h The collected fractions were then analyzed offline by highperformance liquid chromatography with an amperometric detector The detection limit of glutathione was at the picomolar level
To sum up the possibility of online connection with the commonly used analytical instruments is the main advan
tage of microdialysis It enables during surgical operation the in vivo determination of various analytes including oligopeptides Usually no additional sample pretreatment procedures are required However the microdialysis devices used in the methods described in the literature possess rather invasive character and have mainly been applied to the experiments carried out with laboratory animals
Extraction procedures
The low concentration level of analytes usually below the detection limit of commonly used analytical instruments is a significant problem in the case of analysis of oligopeptides from complex sample matrices Therefore the application of supplementary steps for analyte preconcentration is required Unfortunately the preliminary pretreatment stages like homogenization and precipitation as well as membrane separation techniques enable only the isolation and purification of analyte of interest but not its enrichment For that reason in most analyses the application of additional enrichment steps is required to increase the analyte concentration The simultaneous purification and preconcentration of oligopeptides is possible by the application of extraction procedures The classical liquid–liquid extraction and solidphase extraction are most often used
Liquid–liquid extraction
Liquid–liquid extraction (LLE) is the oldest type of extraction technique used commonly in many analytical laboratories Considering oligopeptide analysis liquid– liquid extraction technique has found application mainly in the pretreatment of biological samples (blood plasma and cerebrospinal fluids) [19 35 43] By appropriate selection of the solvent type and pH of an aqueous phase as well as sample ionic strength almost 100 analyte recovery was possible Even though the oligopeptides are polar compounds and extraction into an organic medium is limited the use of an organic solvent or its mixture with some acids usually helps to increase the recovery Selected examples of conditions for liquid–liquid extraction are summarized in Table 2
Liquid–liquid extraction was effectively applied for isolation and preconcentration of synthetic analogs of opioid peptides and substance P from cerebrospinal fluids It helped to remove most of the CSF proteins that interfered with the measurements due to problems with crossreactivity [35] The developed sample pretreatment method highly improved the recovery of neuropeptides at femtomolar concentrations It has been demonstrated that the application of simple liquid–liquid extraction with an organic phase consisting of a mixture of ethyl acetate acetone and petroleum ether enabled the recovery of those analytes with high selectivity Additionally the application of sample acidification with HCl before the liquid–liquid extraction procedure improved the removal of proteins from the sample and significantly increased the enrichment of the analytes For the analysis without HCl about 30–40 higher values of recovery were obtained Compared with the experiments with solidphase SepPak C18 cartridges the application of liquid–liquid extraction increased the enrichment of enkephalin peptides In addition this developed LLE procedure was significantly simpler and more effective than the solidphase extraction (SPE) In this case the analytes were determined at a 2 pgml concentration level
The application of liquid–liquid extraction for isolation and preconcentration of substance Prelated peptides from tissue homogenates and body fluids has been extensively reviewed by Rissler [19] Since those oligopeptides are polar substances their extraction recovery was very low even after performing multiple extractions however the use of various waterimmiscible solvents provided an effective solution A significant increase of analyte enrichment was noticed when an ionpair agent was added (ie TFA) Unfortunately because of the application of mass spectrometry detection in this procedure the removal of inorganic compounds was required
The LLE procedure was also used for the isolation and preconcentration of some cyclic oligopeptide toxins from human body fluids [43] nButanol was successively used for the separation of those compounds from urine samples The total time for liquid–liquid extraction was only 10 min The extraction yields exceeded over 80 In contrast for isolation of these toxins from blood samples the precipitation with acidified acetonitrile followed by centrifugation
Table 2 Selected examples of conditions for liquid–liquid extraction in oligopeptides analysis
Analyte
Sample matrix
Extraction procedure
LODLOQ
Ref
Substance P opioid peptides
CSF
(1) Ethyl acetatepetroleum ether (11)
(2) Acetonepetroleum ether (11)
2 pgml
[35]
Mushroom peptide toxins
Human urine
(3) Ethyl acetate
nButanol
50 ngml
[43]
Substance P
Tissue body fluids
Diethyl ether ethyl acetate methylene chloride hexane
–
[19]
was sufficient The solvent from the extract and the supernatant was removed by rotary evaporation and the samples were analyzed by capillary electrophoresis In both cases the detection limit was about 50 ngml
To conclude despite the simplicity and easy of use of LLE procedures their popularity has decreased in recent years because of numerous disadvantages with this extraction technique eg it is a tedious and timeconsuming procedure that is not easy to automate and emulsion formation is possible during liquid extraction
Solidphase extraction
Solidphase extraction (SPE) is currently the most often used sample pretreatment technique in analytical procedures Compared with liquid–liquid extraction SPE shows lower selectivity but access to many types of sorbents (eg C18 C8 C2 phenyl CN NH2 and ionexchange bonded materials) enables effective and selective isolation and extraction of many classes of compounds The use of reversedphase SPE cartridges makes it possible to moderate and optimize the analyte purification and desalting The addition of small amounts of trifluoroacetic acid to the eluent usually significantly improves the recovery of analytes
Many papers that deal with solidphase extraction applied for oligopeptide analysis have been published recently [7 13 17 40 51 53 62 76–83] In most cases
the complex matrices of analyzed oligopeptides were directly applied to the SPE sorbents and the analytes were then eluted with suitable solutions Sometimes better recovery values were obtained when preliminary precipitating or low pressure chromatographic techniques were used Compared with liquid–liquid extraction methods solidphase extraction provides a more effective sample pretreatment technique for isolation and preconcentration of oligopeptides both from food and biological samples Examples of extraction conditions for solidphase extraction are presented in Table 3
Herreiz and Casal fully reviewed the SPE methods used in the analysis of foodderived peptides [21] Rissler evaluated solidphase extraction procedures applied in the determination of substance Prelated peptides in various types of samples [19] Therefore only some additional examples of SPE application for oligopeptide analysis not included in these reviews will be described below
The octadecyl SPE sorbents were adopted for the quantitative analysis of opioid peptides from human plasma samples [78] The elution was done with a mixture of acetonitrile and trifluoroacetic acid The effluent was then evaporated and the residue was redissolved in a solution of ammonium acetate and analyzed by LC–ESIMSMS The recovery of analytes from plasma was over 80 This method permitted the detection of analytes by utilizing only 025–05 ml of plasma The detection limit of enkephalin peptides in plasma was approximately 1 ngml
Table 3 Selected examples of extraction procedures for solidphase extraction in oligopeptides analysis
Analyte
Sample matrix
Extraction procedure
LODLOQ (ngml)
Ref
Synthetic drug
Human plasma and urine
Sorbent DiolSPE
10
[76]
Cetrorelix
Human plasma and urine
Elution 025 mM NaOH and 1 CF3CO2H in CH3OH
Sorbent C8
2
[13]
Opioid peptides
Human plasma
Elution CH3OH H2O CF3CO2H (901001)
Sorbent C18
1
[79
81]
IRI5 14 (drug)
Rabbit and human plasma
Elution 80 CH3CN01 CF3CO2H
Sorbent C18
2
[53]
Antagonist G
Human plasma
Elution (1) 01 TFA (2) 3 CH3CN in 01 CF3CO2H
Sorbent C2
20
[17]
αAmanitin
Human serum kidney
Elution CH3OH 1 M CH3COONH4
Sorbent C18benzenesulfonic acid
02
[40]
Angiotensin II
and liver
Human plasma
Elution CH3OH
Sorbent CBA
20
[83]
Bioactive
Marine organism
Elution 100 mM CH3COONH4 in 90 CH3OH
Sorbent Amberlite XAD
–
[7]
oligopeptides
Enkephalins
Cerebrospinal fluids
Elution methanolwater
Sorbent C18
100
[77]
Enkephalins
Human plasma
Elution 16 mM ammonium acetate buffer pH 58
Sorbent C18
025
[78]
Cyclic peptides
Rat plasma
Elution 80 acetonitrile01 CF3CO2H
Sorbent C18
6
[80]
Elution 70 acetonitrile30 water
In clinical analysis of peptide drugs highly specific and sensitive methods must be used since limited bioavailability of the drugs often results in low drug levels in plasma Sensitive quantification of the pentapeptide drug IRI5 14 (AcArgProAspProPheNH2) in plasma by using highperformance liquid chromatography coupled with ESIMS has been described in the literature [53] Prior to the analysis the plasma samples were prepared using protein precipitation followed by solidphase extraction The precipitation was used for plasma protein removal whereas SPE C18 sorbent enabled the elimination of the excess of salts and interferents For preconcentration and elution of IRI5 14 a mixture of acetonitrile and 1TFA in water was the most effective The analyte recovery from plasma samples was over 70 The lowest limit of quantification was 2 ngml
Another interesting example of using extraction is the isolation of αamanitin from liver and kidney samples which was possible by the application of homogenization with a mixture of 01 M phosphate buffer (30) and acetonitrile (70) without any analyte loss [40] The obtained homogenate was then centrifuged and the supernatant was shaken with methylene chloride to remove the acetonitrile from the sample Finally the aqueous phase was cleaned up using the solidphase extraction procedure and analyzed by HPLCMS
A microbore analytical column with a C2 sorbent was used for removal of interfering components from blood plasma samples in the case of determination of the anticancer peptide drug (antagonist G) and its three major metabolites [17] The elution of analytes from the SPE column was done with methanol and 1 M ammonium acetate mixture (9010 vv) The limit of quantitation was 20 ngml for antagonist G and 100 ngml for its metabolites
Mixedmode C18benzenesulfonic acid solidphase extraction cartridges were used in a sample pretreatment procedure for the determination of αamanitin in serum and tissues samples (kidney and liver) [40] The SPE procedure was preceded by precipitation in order to remove the interfering substances from the samples The analyte was eluted with methanol evaporated to dryness redissolved in water and injected into an HPLC system Recovery of the analyzed compound was over 98 The detection limit of the developed method was 020 ngml
The solidphase extraction method based on a weak cationexchange mechanism with the use of silica sorbents modified with carboxylic acid (CBA) has been applied for the purification of angiotensin II and gonadoliberins in human plasma [83] Sample impurities were washed off with a mixture of ammonium acetate in water and ammonium hydroxide in acetonitrile Elution took place with a solution of ammonium acetate in methanol Volatile
solvents were irreplaceable for effective removal of undesirable salts from the plasma samples The detection limit for studied analytes was 50 ngml In addition in another work it has been proven that the enrichment factors of analyzed enkephalins from plasma were about 20 higher when C18bonded material was applied compared with a strong cationexchange column (SCX) [80] Unfortunately the high level of salt content in the case of analysis with SCX was unsuitable for the chromatographic instrument used in such experiments
Solidphase extraction methods were mainly used for peptide isolation and fractionation in the case of food sample analysis rather than body fluid samples Usually the high concentration of oligopeptides in protein hydrolysates did not required the use of preconcentration steps Extensive review of the application of SPE methods in food sample analysis has been presented in a paper by Herraiz and Casal [21] and therefore it will not be described in detail Nevertheless it may be concluded that hydrophobic oligopeptides in food are usually extracted with the use of octadecyl (C18) sorbents CN sorbents were adequate for very hydrophilic compounds In the case of nonpolar and hydrophobic analytes the application of a wide spectrum of SPE cartridges such as PH CH C8 and C2 may be useful More details about appropriate selection of sorbents for solidphase extraction of peptides in the case of food analysis may be found in the aforementioned paper
To summarize the solidphase extraction technique is one of the most often used methods in sample pretreatment procedures Unfortunately the use of substantial amounts of organic solvents and the necessity of carrying out many laborious operations are the main disadvantages of this method Furthermore this method is characterized by low selectivity Generally the separation of analytes is based on the hydrophobicity Therefore development of new faster and more selective methods for sample cleanup and enrichment of various analytes from complex matrices is still needed
Membrane extraction techniques
Supported liquid membrane extraction (SLM) may provide an alternative to SPE sample pretreatment methods SLM combines the advantages of classical membrane processes and liquid–liquid extraction offers the possibility to perform separation and preconcentration in one step and uses small volumes of organic solvents (only for making up the liquid membrane)
One method has recently been reported for the SLMbased analysis of short peptides from plasma samples [84] In this procedure SLM was connected online to RPHPLC with UV detection and it enabled the extraction and preconcentration of small model peptides containing from
two to six amino acids including chemotactic (MetLeuPhe) and immunostimulating (ValGluProIleProTyr) peptides in a fully automated system The transport of permanently charged peptides across hydrophobic organic membrane was facilitated by an anionic carrier (Aliquat 336 a quaternary ammonium salt) in the membrane A gradient of chloride anions was a driving force for the process The minimum quantifiable concentration of investigated analytes was in range 90–130 ngml based on 05 ml of plasma The problem of protein interaction with the liquid membrane surface was overcome by using a short ultrafiltration procedure
Low pressure chromatographic methods
A large number of currently available analytical methods for quantification of oligopeptides in complex sample matrices utilize offline sample pretreatment procedures However the online combination of two or more purification techniques is a powerful tool for the determination of peptides Such possibilities may be achieved by the application of low pressure chromatographic techniques including sizeexclusion chromatography (SEC) and ionexchange chromatography (IEC) The use of a small reversedphase cleanup column prior to the application of a mass spectrometry system has also gained interest in recent years The online coupling of at least two techniques in a fully automated system significantly shortens the analysis time and limits possible sample contamination Moreover such a multidimensional system enables the required resolution andor selectivity to be achieved in one step
In the case of sizeexclusion chromatography (SEC) the simultaneous removal of salts and interferents was possible [27 59 85–89] Moreover SEC allowed peptide fractionation that is required in the case of analysis of protein hydrolysates [45 51 62 90–92] Various types of resin like silica crosslinked dextran (Sephadex) and copolymers of divinylbenzene and its sulfate analogues (with various pore size) were used as column stationary phases Depending on the type of resin used oligopeptides were eluted from the column with water organic acids (ie formic and acetic acid) as well as solutions of ammonium acetate or bicarbonate Sometimes the use of a mobile phase that contained alcoholic solutions helped to reduce the potential hydrophobic interaction of the analyte and the silica
Ionexchange chromatography (IEC) permits the fractionation of complex sample components according to the differences in charge This technique was particularly well suited for the isolation of peptides since most peptides possess net charge at side chains or the N or Cterminus The stationary phases commonly used in IEC are resin or
gel matrices that consist of agarose or cellulose beads with covalently bonded charged functional groups respectively Elution of the analyte was usually achieved by increasing the ionic strength of the mobile phase in order to break up the ionic interaction or by changing the pH of analyzed samples There are several reports of selective elution processes in peptide purification [39 93 94] However similar to the case of the application of sizeexclusion chromatography most of them concern the area of food sample analysis [36]
An online system of sizeexclusion chromatography (SEC) and reversedphase liquid chromatography (RPHPLC) was developed for the quantification of structurally related enkephalins in cerebrospinal fluid samples [88] The SEC column was packed with hydrophilically modified silica for the first dimension and C18 for the second A water solution containing glacial acetic acid and ammonium acetate was used as the mobile phase in the first dimension The analytes were eluted under isocratic conditions using acetonitrile as an organic modifier of the mobile phase in the case of the second dimension The limit of detection (LOD) was 5 ngml Unfortunately the analysis of CSE spiked with analytes caused the occurrence of serious interferences between the signals of analytes The increase of the injection volume did not improve the limit of quantification The same SEC conditions were applied for the determination of enkephalin peptides in plasma and cerebrospinal fluid samples [85] After the separation of enkephalins from the sample proteins in the SEC dimension the fraction of analytes was concentrated on the top of the RPHPLC column and finally injected into a capillary electrophoresis system The developed procedure was fully automated Furthermore compared with the earlier described system the quantitation limits of the studied analytes were slightly lower
In the case of a determination of substance P in the rat ventral tagemental area a complex sample pretreatment procedure that involved homogenization extraction and an online system of sizeexclusion chromatography and ionexchange chromatography has been also described [89] The SEC column was packed with Superdex (dextran chains covalently linked to an agarose matrix) Ammonium bicarbonate was used for analyte elution The active fractions were pooled for further separation on a strong ionexchange column (Resource Q) based on rigid beads made of polystyrenedivinyl benzene In this dimension the sample was eluted with sodium phosphate buffer (pH 74) The obtained final fraction of analyte was assayed for SP endopeptidase activity
Sizeexclusion chromatography in an online system with solidphase extraction and capillary electrophoresis has been applied to remove the interfering compounds from a sample of cerebrospinal fluid [77] In these experiments the
application of a SEC column was necessary to eliminate the undesirable proteins present in the samples of cerebrospinal fluids For SEC experiments a highefficiency silicabased gel filtration column was selected For the effective elution of analytes ammonium acetate buffer with pH 58 was utilized The detection limits of the analyzed enkephalins were about 100 ngml
Sephadex G25 column based on crosslinked dextran was used for separation and determination of foodderived oligopeptides in plasma samples [51] In this research the solution of the peptide fraction obtained from the SEC column was then derivatized with phenyl isiothiocyanate to facilitate their specific detection Unfortunately the sizeexclusion chromatography procedure permitted only the removal of plasma proteins from the analyzed samples To improve the resolution of the analytes the solidphase extraction procedure for analyte preconcentration had to be applied The recovery of foodderived peptides was over 70 after this stage The studied analytes were determined at 20 pgl concentration
In the case of food sample analysis the content of peptides compared with biological samples is much higher and an enrichment step is usually not required Therefore the application of only one chromatographic technique for fractionation of sample compounds is sufficient For instance such a method was applied for the purification of antioxidant peptide fractions from mackerel hydrolysates where the use of only sizeexclusion chromatography was required [45] The hydrolysate was fractionated on a Sephadex25 column and eluted with deionized water Three major fractions were obtained The estimated size of these components was 1400 900 and 200 Da The 1 400Da fraction possessed the strongest antioxidant activity
Sephadex25 resin has also been utilized for sizeexclusion chromatography experiments in the case of structural analysis of antioxidant peptides from soybean protein (βconglycinin) A 50 mM acetic acid solution was used for elution of analytes from the SEC column Before the application of mass spectrometry detection the analytes were additionally purified on a semipreparative HPLC column
The Sephadex resin has also found application for the separation of peptides from wine samples This technique enabled simultaneous removal of both small and higher molecular weight interfering components from the analyzed samples Water solutions of acetic acid sodium chloride and aqueous organic solvents mixtures were very useful for peptide elution Furthermore these developed procedures were valid for the analysis of peptides with low molecular weight (<1000 Da)
The use of silicabased strong cationexchange sorbents for purification of some synthetic and proteolytically
derived peptide fragments was described in the literature [93] The sulfoethyl aspartamide resin was utilized as a column stationary phase A gradient elution of acetonitrile and methanol with 85 phosphoric acid was used for IEC The desirable peptide fraction was usually collected at 05to 10min intervals over the entire gradient Nevertheless the application of semipermeable RPHPLC for desalting was necessary in order to perform the mass spectrometry analysis
The work of Sommerer at al [59] is a representative example of the application of a sequential chromatography system to the isolation and purification of cheese peptides In this case the ultrafiltered watersoluble extract was successfully submitted to sizeexclusion chromatography anionexchange chromatography and a mass spectrometry followed by semipreparative reversedphase highperformance liquid chromatography The peptide analytes were eluted with water The IEC fractions were collected manually and after drying under vacuum the samples were dissolved in water and injected onto a semipreparative RP18HPLC column to remove salts from the sample before the mass spectrometry detection Using this method the sequences of 28 short peptides have been obtained
To sum up the application of low pressure liquid chromatography techniques permits mainly fractionation of analytes and removal of sample interferences such as proteins detergents or salts from complex sample matrices Therefore the use of additional sample pretreatment procedures for analyte preconcentration in the case of oligopeptide analysis is required However the disadvantages like significant sample dilution and loss of polar peptides due to their adsorption on the polar crosslinked dextran surface limit the applicability of such techniques
Conclusion
Currently much scientific research focuses on the identification quantification and purification of natural and synthetic peptides The occurrence of peptides in a wide range of samples highlights the need for their analysis in samples of different origin The complexity of the studied material usually requires the application of sample pretreatment techniques for isolation purification and preconcentration of the analyte Combining two or more sample pretreatment procedures usually improves the overall resolution and sensitivity
Examples of sample pretreatment techniques that enable effective and selective isolation andor preconcentration of oligopeptides from complex sample matrices have been reviewed In general the samples of tissue food and biological fluids are first subjected to a preliminary sample cleanup eg homogenization and precipitation to remove
the endogenous interferents (ie proteins lipids and in (organic) salts) from the sample The membranebased techniques and low pressure chromatographic methods for separation and purification of the analytes are then applied The peptide concentration in samples of biological fluids is often at the femtomolar level Therefore the application of extraction procedures for analyte preconcentration is usually required Currently online sample preparation methods and devices tend to be the focus point of research and commercialization
Acknowledgements The authors would like to thank Dr Pawe Dygiel (Institute of Chemistry Opole University Poland) for helpful discussions and suggestions
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