Capillary Electrophoresis Yan Xu Department of Chemistv, Cleveland State University, Cleveland, Ohio 44 1 15
Capillary electrophoresis (CE) continues to be a very active research area in separation science. Compared to the last review period, there has been a significant increase in publications that dealt with the application of CE to clinical diagnosis, biological sample preparation for CE techniques, and CE quantitation. Since the fundamental review of CE can be found in Analytical Chemistry (Nl), the current section will focus on the aspects relevant to clinical application of CE. This is not intended to be a comprehensive review of all published papers in the review period; rather, the author has tried to select those papers that the author feels are significant to clinical chemistry. APPLICATION OF CE IN CLINICAL DIAGNOSIS CE is a powerful separation and quantitation technique that often provides higher resolving power, shorter analysis time, and lower operational cost than high-performance liquid chromatography (HPLC) or conventional gel electrophoresis. According to their charge, hydrophobicity, size, or stereospecificity, a wide range of biologically active molecules such as inorganic ions, organic acids, amino acids, peptides, drugs, nucleosides, nucleotides, vitamins, steroids, hormones, carbohydrates, proteins, and nucleic acids can be separated by CE. Although CE is not a common technique currently used in clinical diagnosis of disease, it has shown great potential for quantitating analytes of interest in biological matrices or profiling metabolites of physiological molecules, which are not routinely analyzed in the clinical laboratory. Profiling. A profile is required when physicians want to evaluate patients’ organ functions or are in search for the cause of definite symptoms of an unknown disease. Profiling usually consists of a multicomponent test in which analytes are structurally or metabolically related. Guzman et al. (N2) and De Antonis et al. (N3)reviewed the use of CE for profiling biochemical molecules in diagnosis of health state and disease. The levels or ratios of certain biochemical markers in a profile can reveal the physiological and pathophysiological states of an individual. Hayes et al. (N4)discussed the prospects of using CE for separation of neuropeptides, catecholamines, and cyclic nucleotides from cytoplasm, organelles, extracellular fluids, and single whole nerve cells. Such studies are essential for eventual understanding of the function of single cells. Weber et al. (NS)developed a CE method to separate the CBI derivatives of the neurotransmitters GAB& glycine, glutamate, aspartate, norepinephrine, and dopamine from 18 other amino acids present in the rat brain. Advis and Guzman (N6) reported a method with fluorescence detection using brain perfusates for determination of in vivo release of multiple neuropeptides (e.g., P-endorphin and neuropeptide Y) from the ewe median eminence. O’Shea et al. (N7)combined microdialysis and CE with electrochemical detection for in vivo monitoring of extracellular levels of aspartate, glutamate, and alanine from the frontoparietal cortex of rat. Deyl et al. (N8) reported the use of CE in profiling of rat hair for alopecia areata diagnosis. Clear differences were observed from both organicand aqueous-phase extracts between hair obtained from alopecia areata-affected laboratory rats and the controls. Lee and Yeung (N9)analyzed the intracellular fluid within single human eryth-
rocyte by CE with laser-induced fluorescence &IF) detection. According to their study, variations among a group of 29 cells in total protein, fraction carbonic anhydrase, fraction Hb &, and an unidentified component were as much as 1 order of magnitude. Although erythrocytes were known to be fairly homogeneous in size distribution, the variations were mainly attributed to the age of erythrocytes. The older cells were less capable of maintaining enzyme activity and preventing oxidative damage. Lal et al. (NlO) conducted a pilot study comparing the salivary cationic protein concentrations of 12 healthy adults controls with those of 12 hospitalized AIDS patients. The authors concluded decreasing histidine-rich protein concentrations, inability of these proteins in saliva to interact with Candida albicans, or both may contribute to the defective salivary antifungal activity in AIDS patients. Bergquist et al. (Nll)used CE with LIF detection to separate and quantitate arginine, glutamine, threonine, valine, y-amino-nbutyric acid, serine, alanine, glycine, glutamic acid, and aspartic acid in human cerebrospinal fluid. Prior to the analysis, these primary amine-containing compounds in untreated cerebrospinal fluid were labeled with 3-(4carboxybenzoyl)-2-quinolinecarboxaldehyde to produce fluorescent isoindoles. The detection limits of these 10 amino acids ranged from 0.29 nM for y-amino-n-butyric acid to 100 nM for threonine. Electropherograms of cerebrospinal fluid samples from patients with Alzheimer’s disease and from children with different neurological disorders were compared to those of health controls. Differences in individual amino acid levels were observed between the patient groups, and these dZferences appeared to be disease and age related. Schmitz and Moellers (NU)developed a capillary isotachophoresis (CITP) procedure for profiling lipoproteins in human serum. The technique was based on prestaining whole serum lipoproteins with a lipophilic dye prior to the separation. Human serum lipoproteins were separated into 14 well-characterized subfractions according to their electrophoretic mobilities. Several genetic disorders of lipid and lipoprotein metabolism such as hyperlipoproteinemias, high-density lipoprotein deficiency syndromes, familial lecithincholesterol acyltransferase deficiency, fish eye disease, hypobetalipoproteinemia, and abetalipoproteinemia were well characterized by the method. Tie et al. (N13)profiled and compared the low molecular weight proteins from human seminal plasma, serum, saliva, and vaginal fluid by CE. According to their study, different types of sample gave different elution patterns which appeared to be applicable for identifying the source of sample. Several papers discussed the separations of isoforms of enzymes such as pig liver esterase isoenzymes (N14), human lactate dehydrogenase isoenzymes (N15,Nl6),Serratia marcescens nuclease isoenzymes (N17), and natural and recombinant fibrinolytic snake venom isoenzymes (N18).The separations of milk proteins by CE were also reported (Nl9, N2O). Screening. A screening test is quantitative measurement of one or a limited number of biochemical markers associated with a known disease state. Tagliaro et al. (N21)published a paper on determination of phenylalanine in serum for diagnosis of phenylketonuria, and Dolnik (N22)reported the separation of pathological metabolites (phenyl pyruvate, 2-hydroxyphenyl acetate, phenyl lactate, and phenyl acetate) from the urine of AnalyticalChemistry, Vol. 67,No. 12, June 15, 1995 463R
phenylketonuric individuals. In both methods, separations were accomplished within 12 min. Currently the analyses are routinely done by liquid and gas chromatography, which require much longer analysis time and more expensive operations. Jansen and de Fluiter ("3) quantitated N-methylnicotinamide (NMN) in rat and human urine by CE, which is considered as a potential biomarker for nitrite exposure. No sample pretreatment was needed prior to the analysis and large series of samples could be handled in their method. Zhu et al. (N24) evaluated the CE separation of abnormal hemoglobins associated with a-thalassemias. Their method could easily differentiate Hbs Bart's and H (associated with a-thalassemias) from Hb variants. Maeda et al. (N25) developed a method for analysis of urinary albumins and Tamm-Horsfall glycoprotein (RIP), which may be a useful marker for patients with renal disease. The separation was accomplished within 12 min. Hiraoka et al. ("6) published a paper on capillary electrophoretic analyses of proteins and amino acid components in cerebrospinal fluid (CSF) from patients with central nervous system (CNS) diseases. The authors found that 1-trace protein was in all the CSF samples examined and its concentration levels were higher in some patients with cerebral infarction and multiple sclerosis. This result suggested that CE may be useful for biochemical diagnosis of CNS diseases. Combining CE technique and the knowledge of molecular biology marked a new era of clinical diagnosis for genetic diseases. Gelti et al. (N27) reported the use of CE to separate polymerase chain reaction (PCR)-amplified DNA microsatellite in a linkage analysis for screening cystic fibrosis (CF) gene in prenatal diagnosis and CF-carrier detection. Nesi et al. (N28) applied CE to detect PCR-amplified genic product for screening Kennedy disease, an X-linked motoneuronal disorder associated with an increase in the number of glycosaminoglycan (GAG), triplet repeats in the first exon of the androgen receptor gene. Khrapko et al. (N29) used a zone of constant temperature and denaturant concentration in capillary electrophoresis for separating mutant from wild-type DNA sequences with high resolution. For a typical 1Wbp fragment, point mutation-containing heteroduplexes were separated from wild-type homoduplexes in less than 30 min. The system used LIF to detect fluorescent-tagged DNA and had an absolute limit of detection of 30 000 molecules. The method had a linear range of 6 orders of magnitude and the percent relative limit of detection was 0.03%. This approach should be applicable to the identification of low-frequency mutations, to mutational spectrometry, and to genetic screening of pooled samples for detection of rare variants. Kuypers et al. (N30) investigated the use of CE in a polymer network for DNA single-strand conformation polymorphism (SSCP) analysis. The PCR-amplified p53 gene (a tumor suppressor gene known to be frequently mutated in malignant cells) was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide were separated. The authors concluded that SSCP using CE in a polymer network can be used for detection of point mutations in DNA sequences. CE technique was also used in determinations of human angiotensin I1 receptor mRNA from the blood of a patient with pheochromocytoma (N31), point mutations at 12th and 61st codons of the N-ras oncogene (N32),and DXS 164locus of the dystrophin gene (N33). Drug Analysis. Clinical drug analysis includes several areas of interest, the most important being (1) therapeutic drug monitoring of specifc drugs or metabolites in blood for assess464R
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ment of organ function, pharmacokinetics, intoxications, and metabolic patterns of pharmacogenetics, (2) screening of drugs of abuse and their metabolites in urine, and (3) athletic drug testing. Currently the high-throughput immunoassay autoanalyzer is the workhorse in clinical drug analysis. About 90%of the drugs of clinical interest are assayed by various types of homogeneous immunoassays. The remaining 10%of the drugs are analyzed by HPLC because of the lack of commercial immunoassays (N34). The limited applications of HPLC in clinical drug analysis are probably attributed to several factors including the following: (1) labor intensive and difficult for total automation, (2) low throughput, and (3) operation experience and periodic troubleshooting required (N35). The advent of CE technique has provided an attractive alternative to HPLC because of its speed, resolution, ease of use, and low operational cost (N34-N39). (A) Athletic and Illicit Drugs. Gonzalez et al. (N40) detected three sport-banned drugs (triamterene, acebutolol, bendroflumethiazide) in human urine by CE with pulsed-laser fluorescence detection. In a separate paper, Gonzalez and Lasema (N41) reported the separation of diuretics, narcotics-analgesics, and 1-blockers. Conditions for the separation of different doping families were discussed in the report, and the analysis of urine from a patient receiving daily doses of atenolol was included. Jumppanen et al. (N42) developed a screening test for diuretics in urine and blood serum by CE with W detection. Diuretics that contained sulfonamide, carboxylic groups, or both were analyzed in 3-(cyclohexylamino)-l-propanesulfonicacid (60 mM) buffer at pH 10.6, and diuretics that contained primary, secondary, or tertiary amine groups were detected in acetate (70 mM)/ betaine (500 mh4) buffer at pH 4.5. All compounds were separated within 30 min and the detection limits were at the low-femtomole level. Chicharro et al. (N43) described a CE method for direct determination of ephedrine and norephedrine in urine. The method had a limit of detection of 2.6 f 0.2 yg/mL for ephedrine and 2.3 f 0.2 yglmL for norephedrine and may be applied for doping control. Tagliaro et al. developed CE methods for determination of cocaine and morphine in the hair of heroin and cocaine users with UV (N44) and fluorometric (N45) detections. The methods were able to detect as little as 0.15 ng/mL of cocaine and morphine in hair using 100-mg samples. Interferences of more than 90therapeutic drugs and drugs of abuse were excluded by the methods. Wemly et al. (N46) reported the analysis of morphine 3-glucuronide by micellar electrokinetic capillary chromatography (MECC), which used Cg cartridges for sample pretreatment and had a limit of detection at about 1yg/mL. (B) Chiral Drugs. Enantiomers are molecules with mirror image structures. There may be differences in physiological effects between enantiomers of racemic drugs. Because stereospecific synthesis of a single isomer can be complex and tedious, a rapid and efficient separation technique for an optically active compound is needed for the pharmacology and pharmaceutics studies. Soini et al. (N47) evaluated the chiral separation of several racemic pharmaceutical bases in both uncoated and polyacrylamide-coated capillaries with modified cyclodextrin derivatives as buffer additives. The optimized separation of the racemic drug bupivacaine was demonstrated in a spiked serum sample at the therapeutic level. According to the authors, the precision, linearity, and sensitivity of the method appeared adequate for reliable quantitation in pharmacokinetic and clinical studies. Aumatell and Wells (N48) developed a CE method for
chiral differentiation of optical isomers of racemethorphan and racemorphan in urine. The method employed a solid-phase extraction procedure for sample pretreatment and a separation buffer containing 60 mM /3qclodextrin, 50 mM SDS, and 20% 1-propanol. It had a detection limit of 20 ppb for urine samples. Francotte et al. (N49) demonstrated the direct separation of some racemic nonsteroidal antiaromatase drugs and barbiturates in plasma samples using CE and MECC. Gareil et al. (N50) determined warfarin enantiomers in the plasma of patients under warfarin therapy by CE with methylated /3-cyclodextrin as the chiral selector. The limit of detection for each enantiomer was 0.2 pg/mL or 0.65pM. Heuermann and Blaschke (N51) reported the enantioselective determination of dimethindene (Fenistil) and its metabolite N-demethyldimethindene in human urine. Dethy et al. (N52) published a CE method for simultaneous determination of verapamil and norverapamil enantiomers in human plasma using trimethyl-/3qclodextrin as the chiral selector. The analysis was achieved within 10 min, and the limit of detection was 2.5 ng/mL. The method had been tested in several thousand human plasma samples, analyzing for the four enantiomers. (C) Drugs in Blood Samples. Shihabi and Constantinescu (N53) quantified iohexol in serum by CE as a measure of the glomerular filtration rate. Comparable results were obtained for serum samples deproteinized with acetonitrile or analyzed directly after %fold dilution with borate buffer. It only took 2.6 min at 12 kV to separate iohexol from other serum components. The separation was performed in a borate buffer, and the detection was carried out at 254 nm with 3-isobutyl-1-methylxanthine as internal standard. Acetonitrile deproteinization gave a greater sensitivity than did sample dilution. Interassay precisions were from 4.7 to 6.7%and intraassay precisions were from 2.5 to 3.2%. Analytical recoveries were between 95 and 105%. Results of the method compared well with those by HPLC. Tomita et al. (N54) applied CE to the determination of six barbiturates in serum. The drugs were extracted from fortifed sera with SepPak Cle cartridges before analyses. Separation was achieved in a buffer containing 10 mM phosphate-borate at pH 7.8, 10%methanol, and 120 mM SDS within 45 min at 20 kV. The calibration curves of each barbiturate had a linear range up to 20 ,ug/mL. Analytical recoveries of six barbiturates were 95.4 and 108.7%at concentrations of 5 and 10 pg/mL. Prunonosa et al. (N55) developed a CE method for the determination of total cicletanine in human plasma. The method included the extraction of the drug with diethyl ether and separation by MECC. This method was compared with a routinely used HPLC method. The CE method required less than a half of the time used by the HPLC method. Shihabi (N56) described a rapid assay for pentobarbital in serum. Serum sample was first deproteinized with acetonitrile and then electrophoresed at 11 kV in 0.3 M borate buffer at pH 8.5. The method demonstrated the potential use of CE for quantitation of small drug molecules in serum. Baillet et al. (N57) used CE with indirect UV detection for determination of fosfomycin in serum. The separation buffer consisted of 200 mM sodium borate and 10 mM phenylphosphonic acid as the W-absorbing background electrolyte. The method was validated over a concentration range (10-100 pg/mL) that is required for therapeutic drug monitoring and used ethylphosphonic acid as internal standard. The interday precision was less than 2%and accuracy was 0.5 and 18%for 100 and 10pg/mL, respectively. Garcia and Shihabi (N58) developed a rapid CE assay for measuring suramin levels in serum. Suramin
is an anti-prostate-tumor drug that has a narrow therapeutic rang and long half-life, and frequent monitoring of the drug is required to minimize its toxicity. In this assay, serum samples were first deproteinized with acetonitrile and then were electrophoresed for 2.5 min at 15 kV in 63 mM CAPS0 buffer (PH 9.7). 3-Isobutyl1-methylxanthinewas used as internal standard and detection was held at 254 nm. The assay had a linear range of 50-500 pg/mL. Johansson et al. (N59)determined theophylline in human plasma by CE. The serum sample was pretreated with acetonitrile to precipitate the plasma proteins. After centrifugation, the supernatant was used for CE analysis. The method had a linear range of 1.8-36 pg/mL (or 10-200 mM), which permits the determination of theophylline in plasma at therapeutic concentrations (4.5-20 pg/mL or 25-110 mM) with acceptable precision. Soini and Novotny (N60) published a paper on quantitative analysis of naproxen, an antiinflammatory drug, in human serum by CE with both W and LIF detections without derivatization. Sample preparation involved a simple step of liquid/liquid extraction. With W detection, ketoprofen was used as internal standard. The linear range was 0.5-25 pg/mL (or 2-100 pM), and the limit of detection was 0.1 pmol. For the low-concentration range (0.010.5 pg/mL or 40 nM-2 pM), LIF detection was employed and warfarin was used as internal standard. The limit of detection was 3 fmol. Schmutz and Thormann (N61) studied the factors affecting the determination of drugs and endogenous low molecular mass compounds in human serum by MECC with direct sample injection. Brunner et al. developed a CE method to detect serum antipyrine, a marker of hepatic oxidative function and total body water (N62). Later, the method was used in the investigation of the effect of individual housing on antipyrine pharmacokinetics (N63).
(D) Drugs in Urine Samples. Tomita et al. (N64) applied CE for the simultaneous separation and detection of nitrazepam and its major metabolites, 7-acetamidonitrazepam and 7-aminonitrazepam, in urine. The three compounds were first extracted from fortified urine with SepPak C18cartridges. The separation was completed within 25 min at an applied potential of 20 kV in a buffer containing 5.1 mM phosphate-borate at pH 8.5, 15% methanol, and 51 mM SDS. The linear calibration ranges were up to 10 pg/mL. The detection limit of each compound was ca. 50 pg (or 0.1 pg/mL) and the recoveries were 78.9-100.8% at 1 pg/mL and 84.1-100.3% at 5 pg/mL, respectively. Zhang et al. (N65) reported the determination of 3-methylflavone-8-carboxylic acid (MFA), the main metabolite of flavoxate, in human urine by CE with direct sample injection. Fenprofen was used as internal standard. The linear calibration range was 1-50 pg/mL and the limit of detection was 0.2 pg/mL, which covered the MFA levels in urine encountered in pharmacokinetic studies. The intraday and interday precisions of the method were less than 2 and 3%, respectively. This method was successfully applied to an excretion study of MFA in eight healthy volunteers, and the results were in agreement with the data in the literature obtained by gas chromatography. Li et al. (N66)developed a CE method for determination of dextromethorphan and its metabolite, dextrorphan, in urine without any sample pretreatment. Linear relationships were observed between the peak area and the concentration of dextromethorphan and dextrophan within the range of 490 ng/ mL to 500 pg/mL with correlation coefficients of greater than 0.9999, The limit of detection was 80 ng/mL for both compounds. The interday precisions at concentrations of 50 and 2.5 pg/mL AnalyticalChemistry, Vol. 67, No. 72,June 15, 1995
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were 4.1 and 6.2% for dextromethorphan and 2.8 and 5.6% for dextrorphan. Using dextromethorphan as the probe of the debrisoquin oxidation metabolic phenotype, 44 healthy volunteers were phenotyped after oral administration of a 15mg dose with the CE method and an HPLC method from the literature. Good agreement was found between these two methods. Arrowood and Hoyt (N67) described the application of CE for separation of cimetidine in rat urine. The method used procaine as internal standard and a liquid/liquid extraction procedure for sample pretreatment. Caslavska et al. (N68) compared the use of three CE methods [MECC, capillary zone electrophoresis (CZE), CITP] for rapid screening and confrmation of drugs in serum and urine of patients with medical drug overdoses (intoxications). Patients' samples obtained from the emergency care unit were analyzed and compared with those obtained by conventional methods. The drugs studied included salicylate, acetaminophen (paracetamol) , and antiepileptics. In the case of high drug concentrations, body fluids could be injected directly or be diluted (urine) or ultrdltered (serum) prior to analyses. Results of analyses were obtained within 30 min. Chee and Wan (N69) reported the separation of 17 basic drugs of different classes in urine and plasma by CE. The method used UV detection and took only 11 min for separation. Tsikas et al. (N70) applied CITP for simultaneous analysis of eicosanoids leukotriene E4, leukotriene B4, prostaglandins E l and E2,f%ketoprostaglandin Fla, thromboxane B2, and their corresponding major urinary metabolites. The method was based on anionic separation and UV detection at 254 nm. Schafroth et al. (N71) published a paper on the determination of flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, oxazepam, and lorazepam in human urine by MECC. The sample pretreatment procedure included enzymatic hydrolysis and solid/ liquid extraction. This method proved to be more sensitive than a commercial EMIT assay. Caslavska et al. (N72) screened hydroxylation and acetylation polymorphism in man via simultaneous analysis of urinary metabolites of mephenytoin, dextromethorphan, and caffeine. No sample pretreatment other than enzymatic hydrolysis of the conjugated compounds was applied. Results of this CE method agreed with those obtained by HPLC. (E) Drugs in Other Biological Fluids. Arrowood et al. (N73) developed a CE method for following the decay of the antibiotic penicillin G or benzylpenicillin in the gastric contents of laboratory rats. The stomach contents was first pretreated by centrifugation and DEAE cellulose and then diluted with phosphate-borate buffer at pH 9 before CE analysis. An internal standard was used to minimize the injection error. The authors concluded that the loss of activity was greater in fasted animals, as expected from the lower pH of their gastric contents, than in fed rats. Perrett and Ross (N74) reported the application of MECC with UV or fluorescence detection for determination of paracetamol and its metabolites in blood plasma and urine, aspirin metabolites in urine, and nucleotides in human tumor cells in fluorouracil-treated rats. Wolfisberg et al. (N75) evaluated the use of CE for determination of bupivacaine in drain fluid collected after pulmonary surgery and MECC for determination of antipyrine in human plasma. Analysis for antipyrine was accomplished without any sample pretreatment whereas bupivacaine required extraction. The data of CE and MECC compared well with the data obtained by gas chromatography (bupivacaine) and HPLC (antipyrine). Gaus et al. (N76)developed a MECC method for separation of primary and secondary cardiac glycosides. The method was applied to 466R
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separate the crude plant cell extracts from Digitalis lanata. Leveque et al. (N77) presented a CE technique for measurement of the antibiotic fosfomycin in serum, cerebrospinal fluid, and aqueous humor. The technique used indirect UV detection and a separation buffer containing organic cation to improve fosfomycin mobility. The separation was accomplished within 7 min. The method had limits of detection of 2.5 and 1pg/mL in serum and aqueous fluids, respectively, and was validated over the concentration range of 2.5-200pg/mL. Tomlinson et al. published a series of papers (N78-N80) on the investigation of the metabolic fate of haloperidol (a neuroleptic drug) in mouse and guinea pig hepatic microsomes by CE and a paper (N82) on the use of nonaqueous solvents in conjunction with the on-line CE/MS for analyzing the hydrophobic drug mifentidine and its metabolites in rat liver microsomes. Endogenous Compound Monitoring. Endogenous compounds include a wide variety of biochemical molecules such as inorganic ions, amino acids, organic acids, carbohydrates, p e p tides, and proteins. Many endogenous compounds are considered as markers and can be used to screen populations, detect the presence of a specific disease, monitor the course of a disease, or predict the prognosis for a patient. The determination of endogenous compounds using CE has been reported by many clinical researchers. (A) Inorganic Ions. Motomizu et al. (N82) reported a CE method for determination of Ba2+,Sr2+,Caz+,and Mg2+. In the method, EDTA was used as the chelating agent in the separation buffer and the separation of these ions was accomplished within 16 min. In another report (N83), they studied the separation of several metal ions with Nfl-bis (2-hydroxybenzyl)ethylenediamineNP-diacetic acid (HBED) as the chelating and coloring agent. Both methods were applied to the determination of Ca2+and Mgzt in serum samples. Luo and Liu (N84) determined K+, Na-, Ca2+, and Mg2+ in blood serum by CITP with a leading electrolyte containing 10 mM HC1 and 0.7%l k r o w n ether and a terminating electrolyte of 10 mM Tris. The results were in agreement with those obtained by ordinary methods with recoveries of 98.0102.5%. Nann et al. (N85) quantitied the K" concentration in blood plasma by CE with an ion-selective microelectrode as on-column detector. The linear calibration range was from to W3M. Zhang et al. (N869 determined ionized and total calcium in human serum by CE with indirect photometric detection. The quantitation was done in a buffer containing 100 mM imidazole (adjusted to pH 7.4 with 5%HzSOJ and 0.1%hydroxypropylmethylcellulose. The results were in agreement with those obtained by the NIST reference method within 95%confidence level. Buchberger et al. (N87) investigated the use of CE with indirect UV detection for determination of low molecular mass cations and anions in serum samples. Janini et al. (N88) applied CE to analyze nitrate and nitrite in urine. No urine pretreatment other than a 4@folddilution was used, and no interference from other anions was observed in the electropherogram. The separation was carried out in polyacrylamide-coated capillary with a modilied phosphate buffer at pH 3, and the detection was at 214 nm. Leone et al. (N89) described a rapid CE method for the measurement of nitrite and nitrate in plasma, which are commonly used indexes for nitric oxide generation. Plasma standard curves gave regression coefficients of 0.98 for nitrite and 0.99 for nitrate, with an intraassay precision of 4.6%for nitrite and 1.2%for nitrate at concentration of 50 pM.
(B) Small Organic Molecules. Koh et al. (N90) measured ascorbic acid (vitamin C) in human plasma samples by CE. Isoascorbic acid, a stereoisomer not normally found in nature, was used as internal standard. The separation was performed in a 30 cm x 75 pm i.d. fused-silica capillary with 100 mM tricine buffer at pH 8.8, and the detection was done by measuring W absorbance at 254 nm. The method had a limit of detection of 1.6 pg/mL, and a linear range up to 480.1 pglmL. The average recovery of the spiked human plasma samples was 98.0%. Xue and Yeung (N91) published a paper on indirect fluorescence determination of lactate and pyruvate in the intracellular fluid of erythrocytes by CE. The authors found that the average amounts of lactate and pyruvate in a single erythrocyte were 1.3 and 2.1 h o l , respectively, or a ratio of 1.6 for pyruvate to lactate. Variations of the absolute amounts and the ratios were fairly large among a group of 27 red blood cells. These were consistent with the differences of cells in size and composition. Hiraoka et al. (N92) described a rapid CE method for determination of lactate in cerebrospinal fluid from patients with central nervous system diseases. Schneede et al. (N93) analyzed methylmalonic acid (MMA) and other shortchain dicarboxylic acids by CE with LIF. The analytes were first derivatized with 1-pyrenyldiazomethane in aqueous matrixes to form stable, highly fluorescent 1-pyrenylmethyl monoesters and then separated in the fused-silica capillary. The LIF detection afforded a limit of detection of about 40 nM for MMA derivatives. Under optimal conditions, the authors were able to detect less than 1pM endogenous MMA in human serum. Wu et al. (N94) separated four major bilirubin species in serum by CE with LIF detection. The separation buffer consisted of 40 mM SDS and 0.012 mM bovine serum albumin (BSA). The use of the SDS/BSA mixture in the buffer allowed the separation of bilirubins at physiological pH with the untreated capillary. The limits of detection were from 30 to 150 nM, depending on the bilirubin species. This method permitted the quantitation of bilirubins in sera without sample pretreatment. Grune et al. (N95) described the separation of adenine, guanine, hypoxanthine, and uric acid in human cord plasma. The method had a linear range of 1-125 pM and a limit of detection of 0.5 pM. In the procedure, plasma samples were first precipitated with an equal volume of HC104 (7%v/v) and then the supernatant was adjusted to neutral pH with potassium carbonate. Prior to the injection, the sample was alkalized with NaOH. The levels of purines were compared with the samples obtained from control newborns, premature babies, and newborns with asphyxia or acidic serum. Ma et al. (N96) presented a rapid assay for serum vitamin A by CE with LIF detection. Subfemtomole levels of vitamin A in human or animal blood were easily detected within 6 min. In a separate paper (N97),Ma et al. reported the indirect photometric detection of several polyamines (putrescine, spermidine, spermine), amino acids (lysine, arginine, histidine), and cations (K+, Na+) by CE. The separation time was less than 10 min, and femtomole amounts of polyamines extracted Erom cultured tumor cells were detected from the nanoliter injection volume. Later, Ma et al. (N98) improved the sensitivity of their previous method by 7-fold, and the improved method was applied to quantitate both bound and free spermidine and spermine in PC12 tumor cells using 1,7diaminoheptane as internal standard. Kristensen et al. (N99) used CE to directly identify and measure the neurotransmitter, dopamine, in two vesicular compartments in a single nerve cell of Planorbis comeus. Dopamine in the cytoplasm and in easily
released transmitter vesicles was separated from dopamine in what were apparently nonfunctional storage vesicles. The two peaks in the electropherogram attributed to dopamine were differentiated by the cell lysis time in a nonphysiological buffer. According to the report, 24% of dopamine was in the cytoplasmic and easily released compartment and 76% was more centrally located, perhaps in a reserve compartment in the cell. This method provided a means for detecting molecular species in subcellular compartments and allowed us to evaluate the kinetic parameters associated with the lysis of a single nerve cell. Lee et al. W O O ) described the determination of creatinine, a useful index of kidney function, in human serum, The method involved deproteinization of 100 pL of serum with acetone, followed by CE separation. The detection limit of creatinine in serum was 0.6 pg/mL. Inflammatory cytokines in pathological tissue samples is an important indicator of disease severity and persistence. Phillips and h e 1 (N101)published a paper on the measurement of tissue-bound cytokines in frozen biopsy specimens by CE. The method used poly(ethy1ene glyco1)coated capillary, performed at neutral pH, and gave consistent and reliable results. (C) Peptides. Soucheleau and Denoroy (N102) developed a method for detecting vasoactive intestinal peptide (VIP) in rat brain. Cerebral cortex was tjrst treated with solid-phaseextraction and purified by reversed-phase HPLC. The VIP-rich fraction was then analyzed by both CE and MECC with W detection. The results of this method agreed well with those obtained by RIA. Yidiz et al. (N103)demonstrated that CE is a preferable technique over HPLC in two examples. The first was the isolation of natural peptides from bovine tissue, and the second was the characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein group 41. Hernandez et al. (N104) discussed the detection of trace amounts of biologically active neuropeptides in tissue and body fluids by CE with LIF detection. Hurst et al. (N105) reported a rapid CE method for the determination of a pentapeptide (enkephalin) in rat retina. The method used ultratiltration for sample preparation, followed by CZE separation with W detection at 210 nm. The analysis time was less than 10 min. Denoroy (N106)developed an electrophoretic method for detecting neurotensin in tissues. Homogenates of rat duodenum and adrenal glands were first extracted by solid-phase extraction and purified by reversed-phase HPLC. The neurotensin-rich fraction was further analyzed by CE with W detection. This approach provided a high level of specificity for analyzing neuropeptides in human tissues. Lim and Sim (N107) presented a protocol for separating 10 peptides of the angiotensin family by CE. This protocol was used to follow the metabolism of angiotensin I in rat lung homogenate and the identification of an angiotensin peptide in human plasma. (D) Carbohydrates and Lipids. Michaelsen et al. (N108) used MECC for the separation and determination of GAG disaccharide units without derivahtion. In their method, samples of chondroitin sulfates and mink skin were first treated with proteases and then chondroitinase ABC prior to the CE separation. O’Shea et al. (N109) coupled CE with a pulsed amperometric detector for direct detection of carbohydrates. The method could detect glucose at the lo-’ M level and was applied to several biologically important carbohydrates. Grimshaw et al. (N110) analyzed glycosaminoglycan hyaluronan in human and bovine vitreous by CE with UV detection. Calibration was carried out AnaIyticalChemistry, Vol. 67, No. 12, June 15, 1995
467A
using standard from known concentrations of hyaluronan of umbilical cord origin. The purity of the standard was examined by lH NMR. A concentration as low as 25 pg/mL was detected by CE. The authors contirmed that the signal was generated by depolymerization of the native mucopolysaccharide via hyaluronidase. The loss of the hyaluronan peak and the appearance of several new peaks corresponding to the oligomeric fragments were seen in the electropherogram. Gangliosides are glycosphingolipids containing sialic acid. These glycolipids have been suggested to play important roles in biological processes such as cell growth, differentiation, and malignant transformation. Based on these proposed biological functions, gangliosides can be used as diagnostic markers and therapeutics for various human diseases. Yo0 et al. (N111)published a report on the determination of several major gangliosides, G M ~G, M ~GDI~, , GDlb, and Gib in mammalian brains. They used a buffer containing 50 mM borate-phosphate and 16.5 mM a-cyclodextrin for separation of gangliosides from extracts of deer antler, apricot seed, and rat brain. (E) Enzymes. Due to their low concentrations in biological fluids, sometime it is difficult to measure enzymes directly by mass. However, enzymes can be measured more easily by their catalytic activities. Bao and Regnier (N112)developed an enzyme activity assay for glucose-&phosphate dehydrogenase using a capillary electrophoretic system. The enzyme was injected by either electrophoresis or siphoning and then mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced NADP, to the detector where it was detected at 340 nm. Under constant applied potential, the migration velocity of enzyme was generally different from that of the product, which was the basis mol of separation. The assay had a detection limit of 4.6 x for glucose-&phosphate dehydrogenase. Pascual et al. (N113) described an assay for glutathione peroxidase activity, which was based on the separation and quantitation of reduced and oxidized glutathione by CE. Landers et al. (N114) measured the activity of bacterial chloramphenicol acetyltransferase (CAT) by CE. In the assay, CAT converted the substrates (acetyl CoA and chloramphenicol) to acetyl chloramphenicol and CoA and CE was used to simultaneously monitor both the loss of substrates and the appearance of products. Miller et al. (N115) assayed leucine aminopeptidase, a clinically significant enzyme, in human serum, human urine, and Escherichia coli supernatant by CE with timeresolved LIF detection. Results of serum and urine samples were within the ranges of expected values found in the literature. Using this method, a concentration of 6 x M enzyme in buffer was detected. Avila and Whitesides (N116) proposed the use of an enzyme plug, migrating in an electrophoretic capillary under nondenaturing conditions, to convert substrate (which was injected onto the capillary as a separate plug or included in the electrophoretic buffer) to product. The concept was demonstrated by two systems: the irreversible oxidation of glucose-&phosphate to Bphosphogluconate using glucose &phosphate dehydrogenase (EC 1.1.1.49) and NAD or NADP as cofactor, and the reversible conversion of EtOH to AcH using yeast alcohol dehydrogenase (EC 1.1.1.1) and N A D 0 as cofactor. The use of CE as microreactor allowed the reactants and products to move in and out of contact with one another on the basis of differences in their electrophoretic mobilities. The proposed concept offered a useful approach to manipulate enzymes and enzyme-catalyzed reactions 468R
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on a microscale. Vinther et al. (N117) used CE to optimize the carboxypeptidase Y (CPDY)catalyzed transpeptidation reaction. A peptide amide (R-Arg-NH2) was produced by CPDY-catalyzed transpeptidation of R-Ala-OH in an excess of Arg-NHz. Baseline separation of R-Ala-OH and R-Arg-NH2 was achieved by CZE. Due to its low sample consumption of the technique, the enzyme reaction was closely followed with an analytical frequency of 3-6/h and the optimization experiments consumed only limited amounts of enzyme and substrate. Mulholland et al. (N118) used CE to assay the activity of tripeptidase from the crude extract of Luctococcus luctis. The assay was able to generate enzyme kinetics data and monitor the presence of coeluting contaminating activities during the puriiication. The speed and sensitivity of CE allowed the routine analysis of tripeptidase activity without any derivatization that was normally required in the other methods. Rogan et al. (N119)used CE to monitor the enantiospecific biotransformation of enantiomers, 2'-deoxy-3'-thiacytidine (BCH 189), catalyzed by cytidine deaminase. The enzymatic reaction was followed by CE over a 51-h time period. The use of dimethy1;Bcyclodextrin as chiral additive in phosphate buffer (PH 2.3) gave enantiomeric separation with results for peak efficiencies, resolution, and analysis time of the order of 25 000 plates, '1.5 and 30 min, respectively. Wu and Regnier (N120) reported the use of CE in gel matrix for conducting enzyme activity assay. Alkaline phosphatase (ALP) and /3-galactosidase were assayed in both C18PF108 modified and polyacrylamide gel-filled capillaries. Enzyme activities were measured by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the enzymatic reaction was monitored by a W/visible detector. Due to the high viscosity of the gel matrix, the polyacrylamide gel column had the advantages of minimized diffusion and limited band broadening. The detection limit for mol (or 7.6 x 10-l2 M). The dual-enzyme ALP was 5.2 x assay of ALP and /?-galactosidase was achieved in a gel-filled capillary simultaneously. Miller and Lytle (N121) investigated the profile of amino acid /3-naphthylamide-aminopeptidaseon the surface of baker's yeast cells (Saccharomyces cerevisiae) by trapping the cells with midcapillary frits. The cells trapped on the capillary were profiled using different enzyme substrates. These substrates were introduced sequentially and incubated for 1min. After incubation, the hydrolysis products were withdrawn by electrophoresis and detected by LIF. An enzyme profile was produced by using only 500 cells. Emmer and Roeraade (N122) combined CE with an on-line postcolumn microreactor for online enzyme assay. Two detectors were employed. The first detector was an on-column UV detector and was used to monitor the CE separation, while the second detector monitored the reaction product which was formed by adding a flow of substrate in the postcolumn section. The separation of glucose-&phosphate dehydrogenase (GGPDH) and &phosphogluconic dehydrogenase (&PGDH) was accomplished by CE where the enzyme activity was monitored after reaction with nicotinamide-adenine dinucleotide phosphate and glucose &phosphate. The minimaldetectable amount of GGPDH was 5 x mol (or 2 x M). Perron and Page (N123) reported a method for monitoring the enzymatic reaction between carboxypeptidase A and methotrexate-a-pep tides. The hydrolysis product (methotrexate) was separated and detected by CE from the substrate. The method may be adaptable for study various enzyme kinetics.
(F') Proteins. Zhu et al. (N124) evaluated the separation of native Hbs and globin chains by capillary isoelectric focusing (CIEF) and CZE. Adult human Hb A, fetal human Hb F, and Hb variants S and C were separated by CIEF via cathodic mobilization, whereas the globin chains of the same Hb species was separated by CZE at low pH in the presence of 7 M urea following the precipitation of the proteins with acidic acetone. Ross et al. (N125) characterized Hb through whole-protein separation and tryptic digest mapping of normal Hb A, common variant Hbs, and some rare Hb variants by CE. In the optimized separation for tryptic digest, up to 28 peaks were resolved in less than 20 min. Ishioka et al. (N126) used CE to analyze the Hb obtained from a male adult Ghanian with retinopathy that was probably caused by hemoglobinopathy. Two major peaks at a ratio of nearly 1 were detected. The elution times of these peaks (Hb XI, Hb XI) were shorter than that of normal Hb (Hb A). Protein sequence analysis revealed that the first peak (Hb XI) was Hb C and the second peak (Hb XI)was Hb S on the electropherogram, and the patient was a heterozygote of Hb S and Hb C (Hb SC disease). This work illustrated that the combined use of CE and protein sequencer can be useful in diagnosis of Hb diseases. Richards and Beattie published a series of papers (N127-N131) on the studies of separation and characterization of methothionein isoforms from livers of different species (e.g., human, rabbit, sheep, and chicken) by various CE techniques. Tadey and Purdy (N132) analyzed the high-density lipoproteins (HDL) and lowdensity lipoproteins (LDL), the main apolipoproteins in plasma, by CE. Using a polyacrylamide-coated capillary, a mixture of HDL and LDL was resolved within 12 min. The preliminary study indicated that CE is a promising technique for screening plasma apolipoproteins. Tie et al. (N133) reported the analysis of human saliva proteins by CE. In their method, saliva samples collected from 32 subjects were analyzed by CE and 8 components (haptoglobin, al-acid glycoprotein, amylase, secretory IgA, IgG, transferrin, prealbumin, albumin) were resolved. Unlike blood serum and seminal serum, the saliva yielded discernible chromatographic patterns from samples of an idnitesimal amount and saliva stains were identifiable after up to 6 months through testing of saliva amylase. Since only a trace amount of saliva was needed for CE analysis, additional forensic and immunological tests was not precluded even if the sample amount was limited. Kim et al. (N134) reported the use of CE for analyzing serum proteins. In their study, serum samples from 38 patients with liver cirrhosis, nephrotic syndrome, or polyclonal gammopathy were analyzed and the results compared well with those obtained by conventional agarose gel electrophoresis and SDS-PAGE. Schmerr and Goodwin (N135) used CE to measure the surface glycoproteins of ovine lentiviruses before and after treatment with glycosidic enzymes. Ovine lentiviruses are a group of viruses that infect sheep and goats. These viruses contain a surface protein, SU, that is very similar among the viral strains. Sera from infected animals react equally well with SU from each strain. Monoclonal antibodies produced against SU can distinguish among some of the viral strains. To delineate the differences, the authors treated SU from several viral strains with the glycosidic enzymes, which included a mixture of exoglycosidases, P-N-acetylglucosaminidase, neuraminidase, and endoglycosidases D, F, and H. After the treatments, the authors observed changes in the reactivities of the monoclonal antibodies that were directed to SU. To characterize these changes on the surface epitopes, SUs from different
Table 1. Selected CE Applications with Sample Pretreatment by Liquidkiquid Extraction analyte
matrix
phenylalanine cocaine/morphine warfarin enantiomers iohexol cicletanine pentobarbital suramin theophylline naproxen antiepileptics cimetidine purine bases/nucleotides creatinine
serum hair plasma serum plasma serum serum plasma serum serum urine cord plasma serum
E : mode
(
CZE CZE CZE CZE
MECC CZE CZE CZE CZE MECC CZE CZE CZE
ref N2 1 N44 N50 N53 N55 N56 N58 N59 N60 N61 N67 N95 NlOO
viral strains were subjected to CE analysis. Differences were readily seen between SU that had not treated and SU that had been treated with the glycosidic enzymes. Each viral strain had a characteristic electropherogram, and the electropherogram indicated that the heterogeneity of the charge on SU was increased after the enzyme treatments. The authors concluded that the carbohydrate moieties play an important role in contributing to the surface charge of SU, and this charge affects the nature of its surface epitopes and has an impact on its biological function. Chen and Stemberg (N136) reviewed the application of CE for protein analyses in biological matrices by untreated fused-silica capillaries. The detection limit of protein, which was based on the intrinsic UV absorbance of the peptide bond at or near 200 nm, was about M. The use of LIF detection could further extend the detection limit to lo-" M. Lerner and Nelson (N137) determined the biological activity and N-terminal sequence of maxadilan, a sand fly salivary gland potent vasodilator, by CE. Goux et al. (N138) analyzed apolipoproteins A-I and A-I1 in human highdensity lipoproteins by CE with a coated capillary filled with a replaceable low-viscosity polymer network containing SDS (eCAP SDS2OO kit from Beckman Instruments). The method used a-chymotrypsinogen A as internal standard; apo A-I and apo A-I1 areas were measured with percent coefficient of variances (%CVs) of 1.6 and 1.8%,respectively. Miura et al. (N139) demonstrated the separation of human serum proteins with CE system. Human serum sample was first diluted 11-fold in 20 mM phosphate buffer at pH 7 and then separated by CE into 10 peaks. The peaks of immunoglobulins, complement C3, transfenin, armacroglobulin, haptoglobin, al-antitrypsin, albumin, and prealbumin were identified. Moreover, the capillary electrophoretic patterns of various patient sera were in complete agreement with the results obtained by cellulose acetate membrane electrophoresis. The authors concluded that the CE technique is suitable for clinical diagnosis. SAMPLE PREPARATION AND QUANTITATION Sample Preparation. The needs and means of biological sample pretreatment for CE analyses were reviewed in a number of papers (N2,N35, N37, N38, N148). When an analyst intends to improve the speciiicity of an analysis or to enhance the mass sensitivity of an assay, a pretreatment procedure is commonly used to remove the interfering components (e.g., proteins) or to concentrate the analyte(s) of interest, or to do both. Many CE applications published in this review period used pretreatment procedures. These methods are summarized in Tables 1-3, which include liquid/liquid extraction (Table 1), solid/liquid extraction (Table 2), and filtration and microdialysis (Table 3). It should also be pointed out that numerous publications reported Analytical Chemisty, Vol. 67,No. 72, June 15, 1995
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Table 2. Selected CE Applications with Sample Pretreatment by SolidAiquid Extraction analyte morphine 3-glucuronide racemethorphad racemorphan isomers barbiturates nitrazepadmetabolites benzodiazepines benzylpenicillin vasoactive intestinal peptide neurotensin y-aminobutyrate
matrix
CEmode
ref
urine urine
CZEMECC N46 MECC N48
serum urine urine gastnc contents rat brain tissues cerebrospinal fluid
MECC MECC MECC CZE CZE CZE CITP
N54 N64 N7 1 N73 N102 N106 N140
Table 3. Selected CE Applications with Sample Pretreatment by Filtration or Microdialysis analyte
matrix
CE mode
low molecular mass seminal plasma proteins salicylate/acetaminophen/ serum antiepileptics amino acids rat frontoparietal cortex rat braidblood phenobarbital glutamate rat brain phenobarbital/ tissues norepinephrine/ amino acids antineoplastic SR 4233/ jugular vein metabolite SR 4317 a
CZE
ref N13a
CZE/MECC/ N68” CITP CZE N7 CZE CZE CZE
N141 N 142-N 144 N145
MECC
N146
Table 4. Selected CE Applications with Direct Sample Injection amino acids N-methylnicotinamide ephedrine/norephedrine aromatase inhibitorharbiturates iohexol antiepileptics 3-methylflavone-8-carboxylic acid
matrix cerebrospinal fluid urine urine plasma serum serum urine
CE mode
ref
CZE
N11
CZE CZE CZE/MECC CZE MECC CZE
N23 N43 N49 N53 N6 1 N65
mephenytoiddextromethorphanl
urine
N66 CZE CZE/MECC/ N68 CITP N72 MECC
caffeine/metabolites antipyrine ionized calciudtotal calcium alkali/alkaline earth metal ions nitratehitrite bilirubins vitamin A proteins proteins proteins theophylline
plasma serum serum urine serum serum saliva serum serum serum
MECC CZE CZE CZE CZE CZE CZE CZE CZE CZE
dextromethorphaddextrophan
salicylate/acetaminophed antiepileptics
urine urine
N75 N86 N87 N88 N94 N96 N133 N134 N139 N153
the use of CE for analyzing biological samples either without any sample pretreatment or only with simple dilution. Some of these methods were listed in Table 4. Quantitation. Quantitative capability is one of the most important characteristics of an analytical technique. Quantitation by CE is still in its early stages of development. The early literature was dominated by the qualitative uses of the technique. Only recently have reviews (N37, N38, Nl47-N149) appeared that addressed the problems of quantitative analysis. This section attempts to give an overview on the subject. 470R
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N38, N147).
(B) Peak Identity and Purity. The simplest method of identifying an electropherographic peak is to compare its migration time with that of a standard. However, as in other separation techniques, the migration time alone is not always reliable to coniirm peak identity and purity. It is necessary to obtain additional information about the peak for final confirmation. One way to achieve this is to compare the ratio of absorbances at different wavelengths between the unknown and the standard in W detection mode or the ratio of currents in potential regions where most changes occur in amperometric detection mode (N150).
Filtration.
analyte
(A) Peak Area and Height. Because peak area is directly related to solute concentration (peak height) and peak residence time (peak width) in the detector, direct comparison of peak area or peak height of analyte resolved by CE with those of calibration standards is possible for quantitative analysis in CE. The reproducibilities of peak area and peak height in CE separation are typically in the order of 1-2% and 3-7%, respectively (N37,
(C) Estimation of Impurity. Atria (N151) discussed the use of peak area normalization for determination of impurity content by CE. The author emphasized that to quantitate the resolved impurity content in the pharmaceutical analysis or directly compare impurity levels between HPLC and CE when results are expressed as percents (impurity area/total area), the use of normalization of CE peak area (peak area/migration time) is essential for ensuring correct quantitation. Because solutes are separated in CE by virtue of differences in their mobilities that cause the differential migration rates of solutes during the electrophoresis (unlike in HPLC, separation occurs via differential partitioning and solutes traverse at the same flow rate), the peak area in an electropherogram is not only proportional to solute concentration (peak height) but also to peak residence time (peak width) in the detector; therefore, direct comparison of peak areas of components resolved in the same electropherogram without peak area normalization may result in severe misinterpretation of the impurity levels. (D) Calibration, Depending on the complexity of the sample matrices, injection mode, and other experimental conditions, either external (direct) or internal calibration graphs can be constructed for the purpose of quantitation. In internal calibration, a known amount of internal standard is added to each calibrator and unknown sample prior to the sample pretreatment procedure. After the sample pretreatment, solution containing calibrator (or unknown) and internal standard is electrophoresed. The peak area (or height) ratios of calibrator and internal standard are plotted against the calibrator concentrations and a calibration graph is constructed. The unknown concentration is determined by knowing the peak area (or height) ratio of unknown and internal standard. Many reports that employed internal standards (N57, N58, N61, N67, N73, N75, N85, N90, N152) showed better assay precisions for a variety of analytes in biological fluids than those of direct calibration. This is because neither the quantities injected nor the detector response needs to be remained constant in internal calibration. (E) Sample Matrix Effect. Sample matrices (N153, N154) strongly influence the quantitative precision and accuracy in CE analyses, especially when electrokinetic injection is used (N155). Leube and Roeckel proposed the use of matrix-corrected peak area (COPA) combined with internal calibration to compensate
the matrix effect on errors in quantitative results in CE with electrokinetic injection (N155). They defined a matrix factor (FM~J as the normalized bias factor that represents the ratio of the peak area (PAJ of analyte i in the sample matrix x vs the peak area (PAJ of analyte i in the calibration matrix c. The matrix factor can be determined either by spiking blank sample matrix with the analyte of interest at a known concentration or by standard addition to biological fluids. Under the condition that Fm, is constant over the entire calibration range, matrix-corrected peak areas (COPAA can be calculated by dividing the peak area raw data (P&J obtained for the sample matrix with the matrix factor The use of COPA instead of peak area (PA) for analyte and internal standard compensates the differences in electrophoretic mobilities between sample matrix and calibration buffer. The authors reported that the intraassay precision and accuracy were improved by factors up to 12 and 6.5, respectively, by using this method; for interassay conditions, the relative deviation was improved from beyond 15%to significantly below 5%from the nominal concentration value. While the separation buffer was kept constant, 12 simulated samples differing in pH, conductivity, and viscosity were tested and the results were satisfying. (F) Reproducibility. Landers et al. (N156) studied the reproducibilities of CE separation for liquid and forced air convection thermostatted systems. Three standards (benzoic acid, peptide from human chorionic gonadotropin, RNase A) representing small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure, respectively, were used for the study. The analyses that were performed in buffers with optimum pHs for the separations demonstrated that both liquid and forced air convection thermostated systems performed extremely well. The reproducibility, as represented by % CV of replicate analyses, was less than 1%of the migration time. The reproducibility decreased in the order of migration time > peak height > peak area. The absolute % CV values with the liquid thermostated system were 2-4fold lower than those observed with the forced air convection thermostated system. However, the difference between the two was not statistically significant. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of increased interaction between analyte and capillary wall. In another study, Waetzig and Dette (N157) also proved that day-to-day precision of below 1%was obtainable by thermosetting. Several papers (N158-Nl60) discussed the factors affecting the reproducibility of CE by either homemade or commercial instruments. Generally, the precisions of these assays were in the range of 1-5%. (G) Limit of Detection and Range of Linearity. Limit of detection is generally dependent on the type of detector used, the chromophore of the analytes, and the conditions of the analysis. For low molecular mass substances and monitoring UV absorption without sample preconcentration, a limit of detection is usually in the low micromolar (or pg/mL) range (N5,N21, N43, N65, N66); if sample is preconcentrated prior to electrophoresis, concentrations as low as a few nanomolar (or ng/mL) in the original samples can be detected (N44,N45, N48, N161). A limit of detection at submicromolar range may be achieved with amperometric detection (N7, N109), and a limit of detection at a few tens of picomolar range is attainable with LIF detection (N136). The linear range of the CE calibration graph has been
as narrow as 1 order of magnitude (N57, N58, N65, N85) or as wide as 6 orders of magnitude (N29). (H)Other Considerations. Spectrophotometric detection in CE may be 1 order of magnitude less sensitive as compared to those available for HPLC because the former has a shorter light passage. This shortcoming can in part be compensated for by the high separation efficiency of CE (the improved resolution resulting in ease of integration), the use of a low UV detection wavelength (down to 185 nm) where many solutes have greater UV absorptivities (N2, N32, N107, N149, N161), and the use of a sample stacking technique (N51, N154, N160) or packed-inlet capillary (N162) for on-column sample preconcentration. Yan Xu is currently an Assistant Professor of Chemistry at Cleveland State University. He received a B.S. degree in chemistryfiom Zhongshan University in 1982. After raduation, he worked as a research chemist in Chinafor three years bejre he moved to the United States. He earned a M.S. degree zn analytzcal chemzstry fiom CalZfornia State Universit Fresno in 1987, and a Ph.D. degree in analytical chemist from t& University of CincinFati in ,1991. DT. Xuk,current reseaJ activities are zn the area of bzoanalytzcal chemzstry wzth focuses on zmmunotechniques and capillary electrophoresis.
LITERATURE CITED Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994,66,280R314R. Guzman, N. A.; Gonzales, C. L.; Trebilcock, M.A.; Hernandez, L.; Berck, C. M.; A d m , J. P. Chromato r Scz Ser 1993, 64 (Capillary Electrophoresis Technologyf ‘643l672: De Antonis, K. M.; Brown, P. R. Am. Lab. (Faifield, Conn.) 1993,25, 62, 64-68, 70-71. Hayes, M. A; Gilman, S.D.; Ewing, A. G. Chromatogr. Sci. &y. 1993,64 (Capillary Electrophoresis Technology), 7537Y9.
Weber, P. L.; O’Shea, T. J.; Lunte, S.M. J. Pharm. Biomed. Anal. 1994, 12, 319-324. Advis, J. P.; Guzman, N. A. J. Liq. Chromatogr. 1993, 16, 2129-2149. O’Shea, T. J.; Weber, P. L.; Bammel, B. P.; Lunte, C. E.; Lunte, S.M.; Smyth, M. R. J. Chromatogr. 1992, 608, 189-195. Deyl, Z.; Tagliaro, F.; Miksik, I. J. Chromatogr., B: Biomed. Appl. 1994, 653, 47-54. Lee, T. T.; Yeung, E. S. Anal. Chem. 1992, 64, 3045-3051. Lal,K.; Pollock, J. J.; Santarpia, R. P., 111; Heller, H. M., Kaufman, H. W.; Fuhrer, J.; Steigbigel, R. T. J. Acquired Immune Defic. Syndr. 1992, 5, 904-914. Bergquist, J.; Gilman, S. D.; Ewing, A. G.; Ekman, R. Anal. Chem. 1994, 66, 3512-3518. Schmitz, G.; Moellers, C. Electrophoresis 1994, 15, 31-39. Tie, J.; Tsukamoto, S.;Oshida, S. Nippon Hoigaku Zasshi 1993, 47, 295-301. Emmer, A.; Jansson, M.; Roeraade, J.J Chromatogr., A 1994, 672, 231-236. Hsieh, Y.-Z.; Chen, F. T. A.; Stemberg, J. C.; Klein, G.; Liu, C. M. U S . 5145567 A, 8 September 1992. Xue, Q.; Yeung, E. S.Anal. Chem. 1994, 66, 1175-1178. Pedersen, J.; Pedersen, M.; Soeberg, H.; Biedermann, K. J. Chromatogr. 1993, 645, 353-361. Markland, F. S.;Moms, S.; Deschamps, J. R.; Ward, K. B. J. Liq. Chromatogr. 1993, 16, 2189-2201. Cifuentes, A.; de Frutos, M.; Diez-Masa, J. C. J. Dairy Sci. 1993, 76, 1870-1875. Chen, F. T. A.; Zang, J. H. J. AOAC Int. 1992, 75, 905-909. Tagliaro, F.; Moretto, S.; Valentini, R., Gamboro, G.; Moffa, M.: Tato. L. Electrobhoresis 1994. 15. 94-97. Dolnik, V. J. Micrololumn Sep. 1994; 6, 63-67. Jansen, E. H. J. M.; de Fluiter, P. J. Liq. Chromatogr. 1994, 17, 1929-1939. Zhu, M.; Wehr, T.; Levi, V.; Rodriguez, R.; Shiffer, K.; Cao, Z. A.J. Chromatogr. 1993, 652, 119-129. Maeda, Y.; Funakushi, IC; Sato, T.; Suzuki, S.; Nakazawa, H.; Ito, K.; Yonekawa, 0. Kanno, T. Shizuoka-ken Eisei Kankyo :,y;m%:Hokoku 1992, 35, 15-19; Chem. Abstr. 1992, 120, I L I JLJ.
Hiraoka, A; Miura, I.; Hattori, M.; Tominaga, I.; Machida, S. Biol. Pharm. Bull. 1993, 16, 949-952. Gelfi, C.; Orsi, A.; Righetti, P. G.; Brancolini, V.; Cremonesi, L.; Ferrari, M. Electrophoresis 1994, 15, 640-643. Nesi, M.; Righe@, P. G.; Patrosso, M. C.; Ferlini, A.; Chian, M. Electrobhoreszs 1994. 15. 644-646. Khra ko, k;Hanekamp, J. S.;Thilly, W. G.; Belenkii, A; Foret, F.; d r g e r , B. L. Nucleic Acids Res. 1994,22, 364-369. Ku pers, A. W. H. M.; Willems, P. M. W.; van der Schans, M. J.; Enssen, P. C. M.; Wesse!s, H. M. C.; de Brui’n, C. H. M. M.; Everaerts, F. M.; Mensink, E. J. B. M. J. hhromatogr., Biomed. Appl. 1993, 62, 149-156. Analytical Chemistry, Vol. 67,No. 12, June 15, 1995
471R
Tomlinson, A. J.; Benson, L. M.; Landers, J. P.; Scanlan, G. F.; Fang, J. J. Chromatogr. 1993, 652, 417-426. Tomlinson. A. 1.:Benson, L. M.; Johnson, K. L. J. Chromatogr., Biomed. App1.-1993, 621. 239-248. Tomlinson, A. J.; Benson, L. M.; Johnson, K. L.; Naylor, S. Elect?pphoresis 1994, 15, 62-71. Tomlinson, A. J.; Benson, L. M.; Naylor, S. J. High Resolut. Chromatogr. 1994: 17, 175-177. Motomizu, S.; Oshima, M.; Matsuda, S.; Obata, Y.; Tanaka, H. Anal. Scz. 1992,8, 619-625. Motomizu, S.; Morimoto, IC;Kuwabara, M.; Obata, Y.; Izumi, K. Bunseki Kagaku 1993, 42, 873-880; Chem. Abstr. 1993, 120, 152464 Luo, Y.; Liu, Fenxi Huaxue 1992,20, 834-837; Chem. Abstr. 1992, 117, 229468r. Nann, ,cc') A.; Silvestri, I.; Simon, W. Anal. Chem. 1993, 65,1662-
Reyes-En el A; Dieguez-Lucena,J. L. Nucleic Acids Res. 1991, 21, 759-966 Baba, Y: Tsuhako, M.; Hayashi, K.; Akashi, M. Kuromatoguraji 1993, 14, 126-127; Chem. Abstr. 1993, 121, 30253. Del Principe, D.; Iampieri, M. P.; Germani, D.; Menichelli, A,; Novelli, G.; Dallapiccola, B. J. Chromatogr. 1993, 638, 277281. Shihabi, Z. K. Ann. Clin. Lab. Sci. 1992, 22, 398-405. Wong, S. H. Y. Am. Clin. Lab. 1993, 12, 27-28,30, 32, 3435. Wong, S. H. Y. Ther. Drug Monit. 1990, 15, 576-580. Thormann, W. Chromatogr. Sci. Ser. 1993, 64 (Capillary Electrophoresis Technology), 693-704. Thormann, W.; Molteni, S.; Caslavska, J.; Schmutz, A. Electrophoresis 1994, 15, 3-12. Evenson, M. A.; Wiktorowicz, J. E. Clin. Chem. 1992, 38, 1847-1852. Gonzalez, E.; Montes, R; Lasema, J. J.Ana1. Chim. Acta 1993, 282, 687-693. Gonzalez, E.; Lasema, J. J. Electrophoresis 1994, 15, 240-243 Jumppanen, J.; Siren, H.; Riekkola, M. L.J. Chromatogr. 1993, 652, 441-450. Chicharro, M.; Zapardiel, A; Bermejo, E.; Perez, J. A.; Hernandez, L. J. Chromatogr., Biomed. Appl. 1993,622,103108. Tagliaro, F.; Poiesi, C.; Aiello, R; Dorizzi, R.; Ghielmi, S.; Marigo, M. J. Chromato r 1993, 638, 303-309. Ta liaro, F.; Antonioli Moretto, S.; Archetti, S.; Ghielmi, S.;%arigo, M. Forensic Sci. Int. 1993, 63, 227-238. Wemly, P.; Thormann, W.; Bourquin, D.; Brenneisen, R. J. Chromato r., Bzomed. Appl. 1993, 616, 305-310. Soini, H.; kekkola, M. L.; Novotny, M. V.J Chromatogr. 1992, 608, 265-274. Aumatell, A.; Wells, R. J.J. Chromatogr. Sci. 1993, 31, 502-
k
IOOI.
Z&n:g,
Xue, Q.; Yeung, E. S. J. Chromatogr. 1994, 66, 287-295. Hiraoka, A.; Miura, I.; Tomina a, I.; Hattori, M.; Sasaki, H. Seibutsu Butsuri Kagaku 1994 38, 233-236; Chem. Abstr. 1994, 121, 174414. Schneede, J.; Mortensen, J. H.; Kvalheim, G.; Ueland, P. M.J. Chromatogr.,A 1994, 669, 185-193. Wu, N.; Sweedler, J. V.; Lin, M. J Chromatogr., B: Biomed. Appl. 1994, 654, 185-191. Grune, T.; Ross, G. A.; Schmidt, H.; Siems, W.; Perrett, D. J. Chromato r. 1993, 636, 105-111. Ma, Y.; u, Z.;.Furr, H. C.; Lammi-Keefe, C.; Craft, N. E. J. Chromato r., Btomed Appl. 1993, 616, 31-37. d a n g , R.; Cooper, C. L. J. Chromatogr. 1992, 608,
6.:
w
Fiancotte, E.; Cherkaoui, S.; Faupel, M. Chirality 1993, 5, 516-.526. Gareil, P.; Gramond, J. P.; Guyon, F. J. Chromatogr.,Biomed. Appl. 1993, 615, 317-325. Heuermann, M.; Blaschke, G. J. Pharm. Biomed. Anal. 1994, 72 753-761) --, . - - . - -. Dethy, J. M.; De Broux, S.; Lesne, M.; Longstreth, J.; Gilbert, P. J. Chromatogr. B: Biomed. Appl. 1994, 654, 121-127. Shihabi, Z. K.; Constantinescu, M. S. Clin. Chem. 1992, 38, 21 17-212n
316. Garcia, L. L.; Shihabi, Z. K. J Liq. Chromatogr. 1993, 16, 2049-2057. Johansson, I. M.; Groen-R dberg, M-B.; Schmekel, B. J. Chromato r 1993, 652, 487-493. ZLni, H.;%ovotny, M. V. J. Microcolumn Sep. 1993,4, 313310.
z h m u t z , A.; Thormann, W. Electrophoresis 1994, 15, 51VI.
L. J.i J.; j DiPiro, J. T.; Feldman, S. J. Chromatogr., Brunner, .L. lj Biome&'Appl. 1993, 622, 98-102. Biomed. Brunnr L. J.; DiPiro, J. T.; Feldman, S . J Pham. Phamacol. Brunner 1994, 46, 581-584. Tomita, M.; Oku ama, T.; Sat0 S.; Ishm, H. Hochudoku 1992, 10, 122-123; Chem. Clem. Abstr. 1992, 1392, 118, 95735k. Zhang, C.; Sun, Z.; Ling, D.; Zheng, J.; Guo, Guo J.; Li, X. J. Chromatnor Rinmrd Appl. Ahhl 1993 612 987-944 Chromatogr., Biomed. 1993, 612, 287-294. L i,s.; Li, S.; IFried, K; Wainer, I. W.; Lloyd, D. K. Chromatographia 1993, 35, 216-222. Arrowc Arrowood, S.; Hoyt, A. M., Jr. Microchem. J. 1993, 47, 9095. Caslavska, J.; Lienhard, S.; Thormann, W. J. Chromatogr. 1993, 638, 335-342. Chee, G. L.; Wan, T. S. M.J. Chromatogr., Biomed. Appl. 1993, 612, 172-177. Tsikas, D.; Fauler, J.; Bracht, S.; Brunner, G.; Froelich, J. C. Electrophoresis 1993, 14, 664-666. Schafroth, M.; Thormann, W.; Allemann, D. Electrophoresis 1994, 15, 72-78. Caslavska, J.; Hufschmid, E.; Theurillat, R.; Desiderio, C.; Wolfisberg, H.; Thormann, W. J. Chromatogr., B: Biomed. Appl. 1994, 656, 219-231. Arrowood, S.; Ho , A M. Jr.; Se aniak, M. J. J. Chromatogr., Biomed. Appl. 1&2, 583: 105-f10 Perrett, D.; Ross, G. Methodol. S U A . Biochem. Anal. 1992, 22, 269-278. Wolfisberg, H.; Schmutz, A,; Stotzer, R.; Thormann, W. J. Chromato r 1993, 652, 407-416. Gaus, H.-f; Treumann, A,; Kreis, W.; Bayer, E. J. Chromatogr. 1993, 635, 319-327. Leveque, D.; Gallion, C.; Tarral, E.; Monteil, H.; Jehl, F. J. Chromatogr., B: Biomed. Appl. 1994, 655, 320-324. 472R
Analytical Chemisty, Vol. 67,No. 72,June 75, 7995
R.; Shi, H.; Ma, Y.J. Microcolumn Sep. 1994, 6, 217-
d
I$,,; "., "".
(N105) (N106) (N107) (N108) (N109) (N110)
Zhang, R.; Cooper, C. L.; Ma, Y. Anal. Chem. 1993, 65,704706. Kristensen, H. K.; Lau, Y. Y.; Ewing, A. G. J. Neurosci. Methods 1994, 51, 183-188. Lee, IC-J.; Heo, G. S.; Doh, H. J. Clin. Chem. 1992,38,23222323. Phillips, T. M.; Kimmel, P. L. J. Chromatogr.,B: Biomed. Appl. 1994, 656, 259-266. Soucheleau, J.; Denoroy, L. J. Chromatogr. 1992, 608, 181188. ,&l% E.; Gruebler, G.; Hoerger, S.; Zimmermann, H.; Echner, H.; Stoeva, S.; Voelter, W. Electro horesis 1992, 13, 683-686. Hernandez, L.; Joshi, N.; Vercfeguer, P.; Guzman, N. A. Chromatogr. Sci. Ser. 1993, 64 (Capillary Electrophoreszs Technolo ), 605-614. Hurst, McLaughlin, P. J.; Zagon, I. S.J. Liq. Chromatogr. 1994. 17. 1877-1881. Denoroy, L. Electrophoresis 1994, 15, 46-50. Lim, B. C.; Sim, M. K. J. Chromatogr., B: Biomed. Appl. 1994, 655, 127-131. Michaelsen, S.; Schroder, M.-B.; Sorensen, H. J. Chromatogr. 1993, 652, 503-515. O'Shea, T. J.: Lunte. S. M.: LaCourse. W. R Anal. Chem. 1993. 65, 948-951. Grimshaw, J.; b e , A.; Trocha-Grimshaw, J.; Douglas, A.; Chakravarthy, U.; Archer, D. Electrophoresis 1994, 15, 936-
8.;
941)
(N111)
(N114) (N115) (N116) (N117) (N118) (N119)
(N125)
?6,Y. S.; Kim, Y. S.;Jhon, G.-J.; Park, J. J. Chromatogr. 1993, 652, 431-439. Baa, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217-224. Pascual, P.; Martinez-Lara, E.; Barcena, J. A,; Lopez-Barea, J.; Toribio, F. J. Chromatogr. 1992, 581, 49-56. Landers, J. P.; Schuchard, M. D.; Subramaniam, M.; Sismelich, T. P.; Spelsberg, T. C. J. Chromatogr. 1992, 603, 247-257. Miller, K. J.; Leesong, I.; Baa, J.; Regnier, F. E.; Lytle, F. E. Anal. Chem. 1993, 65, 3267-3270. A d a , L. Z.; Whitesides, G. M.J. Org. Chem. 1993,58, 55085512. Vinther, A.; Adelhorst, K.; Kirk, 0. Electrophoresis 1993, 14, 486-491. Mulholland, F.; Movahedi, S.; Hague, G. R.; Kasumi, T. J. Chromatogr. 1993, 636, 63-68. Rogan, M. M.; Drake, C.; Goodall, D. M.; Atria, K. D. Anal. Biochem. 1993, 208, 343-347. Wu, D.; Regnier, F. E. Anal. Chem. 1993, 65, 2029-2035. Miller, K. J.; Lytle, F. E. Anal. Chem. 1994, 66, 2420-2423. Emmer, A.; Roeraade, J. J. Chromatogr. 1994, 662, 375-381. Perron, M. J.; Page, M. J Chromatogr. 1994, 662, 383-388. Zhu M: Rodri ez R.; Wehr, T.: Siebert, C. J. Chromatogr. 1992, 608, 22E2-23'7 R0s~z-G.A.; Lorkin, P:; Perrett, D.J. Chromatogr. 1993,636, 6Y-79.
(N126) Ishioka, N.; Iyori, N.; Noji, J.; Kurioka, S. Biomed. Chromatogr. 1992, 6, 224-226.
(N127) Hernandez, L.; Joshi, N.; Murzi, E.; Verdeguer, P.; Mifsud, J. C.; Guzman, N. J. Chromatogr. 1993, 652, 399-405. (N128)1 Hernandez, L.; Escalona, J.; Verdeguer, P.; Guzman, N. A. J. Liq. Chromatogr. 1993,16, 2149-2160. (N129) Roussin, A.; Verdeguer, P.; Hernandez, L. Analusis 1993,21, M42-M45; Chem. Abstr. 1993. 119. 44627a. (N130) Hogan, B. L.; Lunte, S.M.; Stobaugh; J. F.; Lhte, C. E. Anal. Chem. 1994, 66, 596-602. (N131) Moring, S.E. In Capillary Electrophoresis; Grossman, P. D., Colburn, J. C., Eds.; Academic: San Diego, CA, 1992; pp 87in8
(N132)
gi&,A. M., Jr. Chromotogr. Sci. Ser. 1993, 64 (Capillary
Electro horesis Technology), 705-714. (N133) Altna, D. I. Chromatom 1993. 646. 245-257. (N134) Lunte, M. S.i O’Shea, T.3. Electro’ horesis 1994, 15, 79-86. Altria K. D. Chromato ra hia 19$3,35, 177-182. Altria: K. D.; Goodall, Rogan, M. M. Chromatographia 1994, 38, 637-662, (N137) Garcia, L. L; Shihabi, Z. K. J. Chromatogr. 1993, 652, 465-
k.
6. h.;
4GQ
Shihabi, 2. K. J. Chromatogr. 1993, 652, 471-475. Leube, J.; Roeckel, 0. Anal. Chem. 1994, 66, 1090-1096. (N140 Landers, J. P.; Oda, R. P.; Madden, B.; Sismelich, T. P.; Spelsberg, T. C.J. High Resolut. Chromatogr. 1992,15, 517525.
(N141) Waetzig, H.; Dette, C. Fresenius’J. Anal. Chem. 1993, 345, 403-410. (N142) Hettiarachchi, K.; Cheung, A. P. J. Phamz. Biomed. Anal. 1993. 11. 1251-1259. (N143) Coufal, P.;’Claessens, H. A; Cramers, C. AJ. Liq. Chromatogr. 1993, 16, 3623-3652. (N144) Thomas, B. R.; Fan ,X. G.; Chen, X.; T ell, R. J.; Ghodbane, S. J. Chromatogr., Biomed. Appl. l g 4 , 657, 383-394. (N145) Altria, K. D. LC-GC 1993, 11, 438-440, 442.
1:
(N146) Swartz, M. E.; Merion, M. J. Chromatogr. 1993, 632, 209213. (N147) Richards, M. P.; Beattie, J. H.; Self, R. J. Liq. Chromatogr. 1993,16, 2113-2128. (N148) Beattie, J. H.; Richards, M. P.; Self, R. J. Chromatogr. 1993, 632, 127-135. (N149) Richards, M. P.; Beattie, J. H. J. Chromatogr. 1993,648,459468. (N150) Beakie, J. H.; Richards, M. P.J. Chromatogr. 1994,664,129134. (N151) Richards, M. P.J. Chromatogr., 8: Biomed. Appl. 1994,657, 345-355. (N152) Tadey, T.; Purdy, W. C. J. Chromatogr., Biomed. Appl. 1992, 583, 111-115. (N153) Tie, J.; Tsukamoto, S.; Oshida, S.Nihon Univ. J. Med. 1992, 34, 315-323; Chem. Abstr. 1993, 118, 227680a. (N154) Kim, J. W.; Park, J. H.; Park, J. W.; Doh, H. J.; Heo, G. S.; Lee, K. J. Clan. Chem. 1993,39, 689-692. (N155) Schmerr, M. J.; Goodwin, K. R. J. Chromatogr. 1993, 652, 199-205. (N156) Chen, F. T. A.; Stemberg,J. C. Electrophoresis 1994,15, 1321. Lemer, E. A; Nelson, R. J. LC-GC 1994, 12, 36, 38.
[E2Lallemant, Goux, A.; Athias, A.; Persegol, L.; Lagrost, L.; Gambert, P.; C. Anal. Bzochem. 1994,218,320-324.
(N159) Miura, M.; Ui, Y ; Hata, K.; Okabe, H. Rinsho Kagaku (Nippon Rinsho KagadkaG 1994,23,32-37;Chem. Abstr. 1994,121, 174341. (N160) Hutta, M.; Kaniansky, D.; Simunicova, E.; Zelenska, V.; Mada‘ova, V.; Siskova, A J. Radioanal. Nucl. Chem. 1992, 163, 87-98. (N161) Tellez, S.;Forges, N.; Roussin, A; Hernandez, L.J. Chromatogr. 1992, 581, 257-266. (N162) Hernandez, L.; Tucci, S.; Guzman, N.; Paez, X. J. Chromatogr. 1993, 652, 393-398.
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