A C T A U N I V E R S I T A T I S L 0 D Z 1 E N S I S
FOLIA CHIMICA 13, 2004
SAMPLE PREPARATION FOR DETERMINATION
OF BIOLOGICAL THIOLS BY LIQUID CHROMATOGRAPHY
AND ELECTROMIGRATION TECHNIQUES
by Edward Bald
D e p a rtm e n t o f E n viro n m en ta l C hem istry, U niversity o f Lodz, 163 P o m o rsku Str., 9 0 - 236 Łódź, P o la n d
Majority o f the bioanalytical or environmental methods do not use just one chromatografie or electrophoretic step, but rather involve several sample pretreatment steps which sim plfy the matrix, and often preconcentrate and chem ically m odify the analytes. This work surveys typical procedures for sample preparation for most com m only analyzed biofluids with particular em phasis placed on chem ical derivatization o f sulfur amino acids for their determination by liquid phase separation techniques. Recent author's laboratory contribution to the developm ent o f sam ple preparation procedures is merked.
K ey w ords: sam ple preparation, liquid chromatografy, capillary electrophoresis, chem ical derivatization, biological thiols.
1. Introduction
The analysis o f biological samples presents a variety o f problem s which are com m on to high perform ance capillary electrophoresis (H PCE) and high perform ance liquid chrom atography (HPLC). These encom pass: (1) large num ber o f individual com pounds in the sam ple, leading to difficulty in resolving the analytes o f interest, (2) the presence o f com ponents, such as proteins, that can m odify the chrom atographic or electrophoretic colum n, (3) low concentrations o f exo- or endogenous com pounds o f interest, leading to detection difficulties, and (4) conjugation of analytes to proteins and low m olecular com ponents o f the analysed mixture. Taking under consideration the above, it is not surprising that the m ajority o f bioanalytical (environm ental) m ethods do not use ju st one sim ple HPCE or HPLC separation step, but rather involve several sam ple pretreatm ent steps which sim plify the m atrix, and often preconcentrate and chem ically modify the analytes [1-3], Such approach leads to
a relatively purified m aterial being introduced to the final separation unit, m aking the separation sim pler and reliable. There are also several disadvantages of this approach nam ely, m ultiple steps usually require more lengthy and more refined sam ple handling and there is more chance for errors.
The biological fluids that are m ost com m only analyzed are plasm a and urine. W hole blood is less often analyzed, with exception in the case of small anim als and forensic toxicology where this may be necessary. Bile, sweat, tears, milk and saliva can also be analyzed. The ease with w hich sam ples can be analyzed increases with the degree of fluidity, bone being the most difficult to handle and cerebrospinal fluid the easiest.
2. Direct Injection of Biological Fluids
In HPCE there has been considerable interest in perform ing single-step analysis, with direct injection o f body fluids on-capillary. T his is quite often possible because open bare capillaries are less prone to irreversible m odification by sam ple m atrix com ponents than a packed HPLC colum n, and even if adsorption o f som e com pounds does occur, there are few limits on the use of aggressive cleaning procedures. There is very im portant argum ent for the developm ent o f H PC E m ethodology for direct injection analyses. W ith direct injection from m icrocom partm ents within living system s this m icroanalytical technique offers new possibilities for understanding o f a variety o f life processes that could not be studied previously.
Urine and plasm a or serum, the most com m only analyzed biofluids, present different problem s. Urine is generally free of proteins and lipids but contains very many com ponents, concentration o f which depends largely on the diet, and this can account for pushing the resolving pow er o f the separation unit to the limit. Plasm a/serum usually contains fewer individual com ponents at high concentration, but the presence o f a large am ounts o f proteins and lipids constitutes serious challenges. Besides m odification of the capillary walls (in HPCE) or packing material (in HPLC), there is a strong affinity between proteins and analytes, leading to conjugates and direct m easurem ent can miss the total content of the analyte and measure only its free fraction. Thus, chem ical or enzym atic cleavage o f conjugated analytes is still necessary in many cases. The advice to filter the sam ple, if possible, through a 0,45 |xm m em brane to remove any particulate capable o f blocking the capillary or colum n is alw ays valid.
O ne problem with the urine HPCE analysis is the variable, but generally high, concentration o f electrolytes. If limits o f detection are adequate, the urine can be diluted first and then analyzed in moderately concentrated buffer [4J. A nother m eans to m inim ize effects o f matrix com position on resolution and
sensitivity is to use high concentration background buffer [5], This requires very good cooling system or limited electric field strength leading to longer time o f analysis w ithout the loss o f sensitivity. Both capillary zone electrophoresis (CZE) and electrokinetic chrom atography (EK C) have been used for direct analysis o f urine, provided that the analyte concentration is high enough for quantitation by detection system. The main problem is to apply a robust method with adequate resolution o f the analyte from other sam ple com ponents. In EKC with m icellar (M EK C ) or other pseudostationary phases sam ple m atrix effects on the results o f analysis seem to be relatively m inor [6].
The problem s that the high concentration o f proteins in plasm a (about 70 g/1) causes in H PC E are double. They adhere to the wall o f capillary leading to variability o f electroosm otic flow (EOF) and peak-broadening. Secondly, the broad protein peaks m ay interfere with analyte peaks. However, several attem pts of direct injection of plasm a were made. It can be done after several fold dilution of plasm a, provided the background electrolyte is o f high concentration 17). This approach m akes sense only if concentration o f the analyte is high. If undiluted plasm a is injected, vigorous w ashings with strong base or acid betw een runs are needed followed by reequilibration in order to achieve good EOF repeatibility 18], A ddition o f sodium dodecyl sulfate to the washing solution can accelerate removal of proteins and reequilibration [9J. Since a surfactant is present in M EK C by definition, the results of analysis o f directly injected plasm a with this HPCE mode are better than with CZE. In a typical M EKC analysis o f plasm a, the surfactant is strongly bound to the protein, and gives it an overall strong negative charge, which reduces interactions with the capillary surface and causes strong m igration against EOF.
Direct injection o f biological samples into the HPLC system causes colum n blockage due to adsorption or precipitation o f proteins. Plasm a proteins are known to precipitate readily particularly in the pH range 4-8. The signs of colum n deterioration could be such as an increase in colum n back-pressure, and peak splitting or tailing. C olum ns designed to exclude proteins but allow sm aller molecules to interact with the stationary phase are available, although their use is limited.
3. Traditional Sample Preparation Techniques
C onventional sam ple preparation techniques, typical schem e o f which is shown in Fig. 1 are well established and account for the vast m ajority o f HPLC procedures for analysis o f biological sam ples. They also feature widely in the bioanalytical HPCE literature. A relatively large biofluid sam ple is needed, ranging from a several dozen to a few milliliters. After preparation, a few
m icroliters to a few m illiliters are available for a final analysis. T he final analysis could be done by HPCE or HPLC. C onsidering small volum e sam ple requirem ents o f H PC E its m icroanalytical abilities are not utilized. The justification for using capillary electrophoresis as the final step o f the analytical procedure may be its excellent resolving capabilities for analytes which could be difficult to separate by HPLC, e.g. diastereoisom ers. As show n in Fig. 1 the principal steps in sam ple preparation are extraction, deproteinization, and chem ical m odification.
Standalone On line
Manual Automatic
Standalone On line Automatic Automatic
standalone on line
Fig. 1. Main steps in the conventional sam ple preparation procedure
Both liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are in use. In LLE, an im m iscible organic phase is added to the biological fluid and these are then shaken together, resulting in the m ore hydrophobic sam ple com ponents being extracted into the organic phase. In SPE, the sam ple is passed over a particulate material packed into small disposable colum ns. Some com ponents are retained on the colum n, and weakly sorbed com ponents may be rem oved by rinsing, follow ed by elution o f strongly retained com pounds. In both LLE and SPE the analyte concentration is possible by evaporation o f solvent and reconstitution o f the residue with small volum e o f desired liquid. T his liquid could be the HPCE background buffer or HPLC m obile phase. Extraction can be done with some degree o f specificity.
M any deproteinization m ethods have been described |10] and the more com m only em ployed are (1) precipitation by addition o f organic solvents, (2)
Deproteinization
Organic Strong Dialysis
solvents acids Chemical modification Reduction Derivatization oxidation blocking Manual Automatic
precipitation by form ation o f insoluble salts, (3) ultrafiltration, and (4) dialysis. U ltrafiltration has an advantage over protein precipitation and dialysis in that it is applicable to small volumes and no dilution occurs.
Since there are opportunities for loss o f analyte at each step in an off line procedure several workers have been looking for clean-up and concentration procedures for HPCE either on-capillary, or using a m iniaturized system coupled to the capillary. A variety o f techniques can be used including liquid chrom atography, electrophoresis and m icrodialysis or ultrafiltration [11].
C hem ical m odification encom passes chiefly derivatization o f the analyte and decom position o f its conjugates with proteins and low m olecular endo- or exogenous com pounds.
4. Thiols as an Analytical Objccts
Thiols play a specific role am ong reduced sulfur com pounds; they are chem ically and biochem ically very active com ponents o f the sulfur cycle o f the natural environm ent and have been extensively studied in various biological system s. Many biological phenom ena, am ong others, redox-, m ethyl transfer-, and carbon dioxide-fixation reactions, are believed to be dependent on the presence o f a thiol group. The determ ination o f thiol containing com pounds is im portant for biochem ical research, in pharm acodynam ic studies o f the thiol drugs, or in the diagnosis o f several diseases, e.g. cystinuria and hom ocystinuria. T he hom ocysteine assay is a sensitive tool for early diagnosis o f disturbed rem etylation and transsulfuration leading to hyperhom ocysteinem ia, which in turn is associated with an increased risk o f atherothrom botic vascular events
[12].
The analysis o f thiols can be quite perplexing. A side from the great susceptibility to oxidation, which can occur before or during analytical process, most thiols lack the structural properties necessary for the production o f signals com patible with com m on HPLC or HPCE detectors, such as UV absorbance and fluorescence. Therefore, the analyst must resort to derivatization for signal enhancem ent and labile sulfhydryl group blocking if fluorescence or UV-Vis detection m ethods are em ployed. Num erous reagent are available for the derivatization via-SH group and subsequent HPLC or H PC E analysis. A m ajority o f the reagents can be classified by type o f the reactive m oiety into three categories: activated halogen com pounds, disulfides, and com pounds Possessing m aleim ide m oiety, and are review ed with som e experim ental details >n som e excellent w orks [2, 13]. However, increased dem and for m easuring thiols, m ostly biological ones in clinical practice, raises the issue o f developing
new m ethods better suited to accom m odate high testing volum es and faster turnaround time.
5. Chemical Derivatization of Thiols for HPCE and HPLC
W e have developed in our laboratory several thiol specific derivatization reagents belonging to the class o f azaarom atic com pounds with an active halogen atom. These are 2-halopyridinium [14] and 2-haloquinolinium salts [15], which react rapidly and quantitatively with hydrophilic thiols in slightly alkaline w ater solution to form stable thioethers, S -pyridinium and S-quinolinium derivatives, respectively. Fig. 2. Shows the derivatization reaction schem e for thiols represented by hom ocysteine [16] with the use o f 2 -c h lo ro -l- m ethylquinolinium tetrafluoroborate (CMQT).
Fig. 2. Chem ical derivatization reaction equation o f hom ocysteine with 2 -ch lo ro -i- m ethylquinolinium tetrafluoroborate
This derivatization schem e takes advantage o f great susceptibility o f the quinolinium m olecule at 2 position to nucleophilic displacem ent, and the high nucleophilicity o f the thiol group. The reaction is accom panied by an analytically advantageous bathochrom ic shift from reagent m axim um (328 nm) to the m axim um o f the derivative (348 nm). This phenom ena is displayed in Fig. 3. O f different functionalities (e.g. -COOH, -N H k -SH) o f hydrophilic thiols potentially able to undergo nucleophilic attack at the 2-position o f quinoline (pyridine) m olecule, only the sulfhydryl group reacts. T his m eans that no m ultiple derivatives are formed. Under the recom m ended derivatization conditions, form ation o f the 2-S-quinolinium derivatives is over within 1 min, that is virtually ju st after m ixing substrates.
Based on 2-halopyridinium and 2-haloquinolinium salts as derivatization reagents we were able to elaborate several m ethods for determ ining endo- [14,16-21] and exogenous [20-25] thiols in plasm a and urine by manual [14,16-25] as well as, fully autom atic [26] manner.
The bulk o f plasm a thiols occur in the disulfide form s rendering them inaccessible to derivatization reagent, and in order to determ ine their total contents disulfide bonds must be cleaved with suitable reducing reagent to liberate a free thiol. For this purpose sodium borohydride [16],
tri-n-butylphosphine [18,24] or 2-m ercaptoethanol [19] was used. Total content o f thiol is understood as the sum o f reduced thiol, free oxidized (dim er and mixed disulfides with other endo- and exogenous thiols) and protein bound thiol.
Fig. 3. Three-dim ensional chromatogram o f the derivatization mixture m ade with continuous spectral scanning during the elution. Peaks from right: glutathione - C M Q T, hom ocysteine - CM QT, cystein e - CM QT, cystein ylglycin e - CM QT derivative and CM QT excess. A x is titles: X - tim e, Y - w avenlenght and Z - absorbance
A m ong plasm a thiols, hom ocysteine is the m ost often target o f analysis. Elevated concentration o f total hom ocysteine in plasm a (hiperhom ocysteinem ia, concentration above 15 nm ol/m l) is recognized as an independent risk factor for prem ature developm ent o f cardiovascular diseases. H ow ever, there is an evident need to m easure different species o f thiols in order to fully understand the dynam ic relationship betw een hom ocysteine and other biologically im portant thiols and disulfides in plasma. Alteration o f the redox status o f hom ocysteine rapidly affects and is related to the total thiol redox status in plasm a. This observations can be explained by continuos redox and disulfide exchange reactions in plasm a. T he com prehensive m easurem ent o f the reduced thiol fraction has proven to be problem atic because o f low concentration and susceptibility to oxidation. T his prom ted us to develop a new H PLC m ethods with UV detection for determ ining different species o f thiols in plasm a [16,17] and urine [27]. T he m ethods rely on transform ation o f thiols to stable 2-S -quinolinium derivatives in the reaction with C M Q T reagent, and separation and quantitation by ion-pairing reversed-phase HPLC. O xidized species are converted to their thiol form s by reduction before derivatization. T o circum vent the loss o f reduced form ex vivo due to oxidation, the C M Q T reagent is added to w hole blood im m ediately after collection and prior to separation o f plasm a from erythrocytes in order to block the reactive -SH groups. T ypical chrom atogram s
for standard m ixture and reduced fraction o f main plasm a thiols are shown in Fig. 4.
A nother new HPLC m ethod (Fig. 5) enables sim ultaneous determ ination of N -acetylcysteine and four main endogenous plasm a thiols [25]. N -acetylcysteine is adm inistered orally or injected intraperitoneally in order to increase the free radical scavenging pow er of hum an plasm a or in the capacity o f mucolytic agent. 119 -f-99 < 79 + £ I 59 ■' o £ 39 --< 19 -I ■ t " 'V P ■t— t* -I 1 3 5 7 T i m c | m i n | —I 9 II T i m e | i n i n |
Fig. 4. Typical chromatogram o f main plasma thiols. (A ) water standard solution, concentration o f analytes and internal standards in final analytical solution 20 nmol/ml in respect to each; (B) reduced thiols in plasma. Peaks: I, glutathione - CM QT 2.51 nmol/ml; 2, hom ocysteine - CMQT 0.31 nmol/ml; 3, 2-m ercaptopropionic acid - CM QT (internal standard) 3.33 nmol/ml; 4, 3-m ercaptopropionic acid - CM QT (internal standard) 3.33 nm ol/m l; 5, cystein e - CM QT 12.1 nm ol/m l; 6, cystein ylglycin e - CM QT derivative 2.36 nmol/ml; 7, ex cess o f CM QT
19 3 14 1 o 2 9 B ■e0 1 4 -I 0 2 4 6 8 10 12 Time [min]
Fig. 5. Typical HPLC chromatogram o f human plasma spiked with 4 0 nmol/ml o f N -acetylcystein e and 4 0 0 nm ol/ml 3 ,3 ’-dithiodipropionic acid (internal standard) and derivatized with 2-chloro-1-m ethylquinolinium tetrafluoroborate (CM QT). Peaks: 1, S-quinolinium derivative of N -acetylcysteine; 2, S-quinolinium derivative o f glutathione; 3, S-quinolinium derivative o f hom ocysteine; 4, S-quinolinium derivative o f 3-m ercaptopropionic acid; 5, S-quinolinium derivative o f cysteine; 6, S-quinolinium derivative o f cystein ylglycin e; 7, ex cess o f CM QT
6. Conclusions
The m ajority o f bioanulytical m ethods do not use ju st one sim ple 11PCE or HPLC separation step, but rather involve several sam ple pretreatm ent steps which sim plify the m atrix, and often preconcentrate and chem ically m odifity the analytes. HPCE follow ing extensive clean - up involves 110 less effort than perform ing an HPLC separation, but for certain applications, such as chiral separations, there are clearly attractions in using HPCE. M inim al preparation by removal o f proteins is satisfactory, provided that the analyte concentration is within the useful range o f the detection system in use. In the case o f biological thiols, for the reason o f their susceptibility to oxidation and lack the structural properties necessary for the production of signals com patible with com m on HPCE or HPLC detectors, derivatization is alm ost always perform ed.
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[17] E. Bald, G. Chwatko, R. G łow acki, K. Kuśmierck, J. C hrom atogr. A, 1032 (2004) 109. [18] E. K aniowska, G. Chwatko, R. G łow acki, P. Kubalczyk, E. Bald, J. C hrom atogr. A, 798
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[21 ] E. Bald, R. G łow acki, J. Drzcw oski, J. C hrom atogr. A , 913 (2001) 29. [22] S. Syp niew sk i, E. Bald, J. C hrom atogr. A, 729 (1996) 335.
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[26] E. Bald, F ully a u to m a ted H P L C b a se d ana lysis o f cystein e a n d rela te d c o m p o u n d s in p la sm a using on line m icro d ia ly sis a s sa m p le p rep a ra tio n , 7th International C ongress on A m ino Acids and Proteins, August 6-10, 2 0 0 1 ,Vienna, Austria, Am ino A cids, 21 (2001) 3.
[27] R. G łow acki, A. Kropiwnicka, J. D rzewoski, E. Bald, D eterm in a tio n o f R ed u ced and O xid ized S p ecies o f C ysteine a n d H om o cystein e in H um an U rine b y H P L C with U ltraviolet D etectio n , H om ocysteine M etabolism , 3rd International Conference, Sorrento, 1-5 July 2001, Abstracts B ook, 128.
