High-Performance Liquid Chromatography in Clinical Analysis

David J. Anderson was the overall coordinator of the clinical chemistry review. He received his B.S. in chemistry from Bucknell University in 1977 and...
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Anal. Chem. 1999, 71, 314R-327R

High-Performance Liquid Chromatography in Clinical Analysis David J. Anderson

Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115 Applications of high-performance liquid chromatography (HPLC) in clinical analysis are critically reviewed in the present article for the Chemical Abstracts time period from October 1, 1996, to October 1, 1998. The present review focuses on those articles using HPLC for clinically relevant analytes, i.e., HPLC of disease markers pertinent to disease diagnosis, treatment, and/or prognosis of disease. The present review is divided into five subsections. The first subsection covers reviews, books, and invited papers, classified according to analyte class. The second subsection discusses references that are grouped according to clinically significant analyte classes, including the following: hemoglobin variants and glycated hemoglobin, vitamins, lipoproteins, nitric oxide metabolites, glycoproteins as disease markers, DNA/RNA, and other disease markers. Criteria for choosing significant articles in this subsection include the following: papers reporting extensive profiling capabilities (reporting separation of many compounds), evaluations of techniques that are either extensive or particularly applicable to the clinical laboratory, reports utilizing HPLC analysis for new or upcoming markers, works in which HPLC is shown to have an advantage over other techniques, reports of reference or potential reference methods, and/or works showing other indications of significance. The third subsection selects papers that show innovation or characterization of the separation mode, with applicability to clinical analytes. The fourth subsection cites significant sample preparation articles. The fifth and final subsection covers HPLC detection methods, citing the following: reviews; significant articles in clinical analysis focused on the improvement in, characterization of, or novel employment of HPLC detection methodology; immunodetection articles; and articles reporting femtomole detection limit capability. HPLC analysis of proteins and peptides, including the coupling of HPLC to mass spectrometry, is a very important application area of HPLC to bioanalysis. This area is covered by citing reviews or invited papers (see Tables 7 and 8) and for cases directly related to clinical analysis. The reader is referred to another review article in the present Application Reviews issue of Analytical Chemistry for a more extensive discourse beyond this focused scope. BOOKS, REVIEWS, AND INVITED PAPERS A previous Application Reviews article covers the use of HPLC in clinical analysis from 1994 to 1996 (D1). A book covering the principles and practice of HPLC and capillary electrophoresis has been published (D2). Books on HPLC applied to bioanalysis include the use of HPLC in measuring enzyme activity (D3) and in pharmaceutical analysis (D4). A substantial number of HPLC reviews have been published for a particular analyte or class of 314R Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

analytes relevant to clinical or biomedical applications, as given in Table 7. Invited papers and chapters from Methods in Enzymology, Methods in Molecular Biology, and other sources are given in Table 8. CLINICALLY RELEVANT ANALYTES Hemoglobin (Hb) Variants and Glycated Hemoglobin. A cation-exchange HPLC technique has been published separating 40 of the most frequently occurring Hbs and Hb variants within 12 min, separating some variants that could not be resolved by conventional electrophoretic techniques (D151). Another work used the Bio-Rad Variant analyzer (which is an automated cationexchange HPLC technique), characterizing the retention time of 125 variants (D152). This work proposed to use this HPLC technique to identify the ambiguous cases in electrophoretic analysis (10% of total, as estimated by the authors). Other work employing the Bio-Rad Variant instrument includes validation of the assay for HbS quantification (D153) and modification of the technique to quantify Hb Bart’s (D154). An HPLC/electrospraytandem mass spectrometry (HPLC/ESI-MS/MS) technique shows promise for identifying the exact substitutions of amino acids in variants by the analysis of enzymatically digested oxidized globin subunits, as demonstrated by the identification of a single substitution in the Hb (classified as Hb Santa Ana) of a patient with congenital hemolytic anemia (D155). Hemoglobin A1c (HbA1c) assays are used for assessing the longterm regulation of blood glucose in diabetics. Although the analysis of HbA1c by ion-exchange HPLC is for the most part reliable, a periodic problem is the coelution with HbA1c of certain Hb variants and adducts. For example, Hb-Raleigh was shown to coelute with HbA1c on the Bio-Rad Variant system, leading to a falsely elevated HbA1c (46%) (D156). The interfering Hb-Raleigh was determined to be an acetylated form of the variant, with the acetylation shown to occur at the substituted N-terminal valine of the β-chain. Comparison of two HPLC analyzers (BioRad Variant and Hi-Auto A1c) and the Roche immunoassay method with the Bio-Rad Diamat HPLC technique shows good concordance of all the techniques in the determination of HbA1c (D157). However, this study showed HPLC was not suitable for patients having Hb variants (at least for those examined). A Mono S cation-exchange FPLC method separated several minor peaks that would normally be included in HbA1c analysis (D158). In this study, uremic patients were shown to have elevated HbA1c (+0.8% units for nondiabetics), which is attributable to increases in several of these minor peak fractions (D158). These minor peaks were initially characterized in an earlier work (D159). 10.1021/a1999904m CCC: $18.00

© 1999 American Chemical Society Published on Web 05/20/1999

Table 7. Reviews of HPLC Techniques for Clinically or Biomedically Relevant Analytes class/subclass of analyte amino acids D-amino acids tryptophan and metabolites (free and hydrolysates of peptides and proteins) arachidonic acid metabolites prostaglandins bile acids carnitine and acylcarnitines carbohydrates sugars oligosaccharides separation and detection techniques sensitive detection techniques conjugate analysis glycoconjugates glycoproteins (see proteins also) glycosaminoglycans (heparin and heparan sulfate) DNA, nucleic acids DNA, double-stranded, micropellicular packing material DNA, oxidized bases oligonucleotides, synthetic drugs antibiotics general cephalosporins, third generation fluoroquinolones macrolides (erythromycin and related substances) β-blockers coumarins serotonin reuptake inhibitors detectors LC/MS, LC/MS/MS, drug metabolism LC/NMR, drug metabolism free radicals, their reaction products, antioxidants inorganic ions cations in serum by ion chromatography sulfate and thiosulfate in clinical samples by anion-exchange chromatography lipids general lipids, preparative HPLC polar lipids, LC/MS/MS carboxylic acids, fluorescent derivatization fatty acids and triacylglycerols, reversed phase phospholipids lipoproteins metals speciation, ion chromatography, atomic and mass spectrometic detectors metallothioneins in cytosol, LC/AAS, LC/ICP/AES, LC/ICPMS, LC/ESI-MS/MS platinum and gold in drugs nitric oxide metabolites nitrite and nitrate S-nitrosothiols peptides general peptides, synthetic separation mode various size exclusion new stationary phases, electrochromatography, peptide drugs a

refs D5a D6a

D7 D8a D9,a D10a D11a D12 D13a D14a D15 D16,a D17a D18 D19a D20a

D21 D22 D23 D24a D25a D26a D27,a D28a D29 D30 D31 D32 D33

D34 D35 D36 D37 D38, D39a D40 D41 D42a D43a D44,a D45,a D46 D47a D48 D49

class/subclass of analyte peptides (contd) detection derivatization mass spectrometry, LC/ESI-MS specific peptides β-alkyl amino acids and peptides containing them MHC-associated peptides, LC/ESI-MS/MS microcystins neuropeptides enkephalins and related peptides methionine enkephalin and β-endorphin in pituitary, by LC and FAB MS opioid neuropeptides by RP-HPLC profiling peptides in development and neurological diseases proline-containing peptides proteins general proteins and hormones separation mode ion exchange, proteins and macromolecules reversed phase, proteins and polypeptides new packing materials detection labeling for improved detection/separation, preconcentration methods mass spectrometry LC/ESI-MS LC/ESI-MS/MS proteolysis tryptic digestion, reversed phase posttranslationally modified proteins glycoproteins (see also carbohydrates) oligosaccharides of IgG, patients with rheumatoid arthritis N-linked glycans from electrophoresed proteins separation methods, HPAEC-PAD phosphorylation other modified proteins adduct markers intact modified proteins unstable proteins, procedures for keeping in native form specific proteins allergens angiotensin-converting enzyme cytochromes P450 and b5 hemoglobins and their chains hemoglobin adducts proteins and peptides proteins, peptides, amino acids nonporous silica packings, short columns pyridinium cross-links pterins quantitation of tetrahydrobiopterin enzymes vitamins ascorbic acid, dehydroascorbic acid, and metabolites, ion-pairing HPLC, multiwavelength UV and electrochemical detection carotenoids thiamin

refs

D53, D54a D55 D56 D57a D58 D59a D60a D61 D62 D63 D64 D65 D66 D67 D68 D69a D70a D71, D72a D73

D74 D75 D76a D77a D78a D79a D80 D81a D82a D83 D84 D85a D86a D87 D88 D89 D90

D91 D92

D50 D51 D52a

Multiple techniques covered, including HPLC.

More specific HPLC techniques are given below to address these interference concerns in HbA1c determinations. A reference method has been proposed for determination of HbA1c, in which enzymatic cleavage of HbA1c (and HbA0) was performed to produce β-N-terminal hexapeptides, which were then subsequently determined by reversed-phase HPLC/ESI-MS or CE (D160). Evaluation of the Menarini-Arkray HA 8140 HbA1c system (a highly automated HPLC system which includes bar code reading,

cap piercing, and whole blood sampling) was performed (D161, D162). Common Hb variants (S, C, F) were shown not to interfere, while the procedure removed a potentially interfering labile Schiff base adduct (D161). Hb variants were resolved from HbA1c on the HA 8140 analyzer, but not on the Bio-Rad Diamat or HA-8121 Menarini HPLC when standard methods were used (D162). A multilaboratory study was done (119 laboratories, 12 methods) showing the utility for using HbA1c calibrators (analyzed by four Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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Table 8. Invited Papers and Chapters of HPLC Techniques for Clinically or Biomedically Relevant Analytes class/subclass of analyte amino acids derivatizations, precolumn and postcolumn PTH amino acids phenylisothiocyanate derivatization, reversed phase arachidonic acid metabolites leukotrienes and other lipoxygenase products carbohydrates monosaccharides released by exoglycosidase, HPAEC-PAD oligosaccharides comparison of 2 derivatization and 4 separation methods HPLC and HPAEC, oligosaccharides and glycopeptides three-dimensional HPLC, N-linked oligosaccharides profiling and sequencing HPAEC, sample preparation, 2-aminobenzamide derivatization 2-aminobenzamide and anthranilic acid derivatization, mono- and oligosaccharides biotinylated diaminopyridine derivatization amylodextrins, HPAEC-PAD, postcolumn enzyme reactor DNA, nucleic acids synthetic oligonucleotides, phosphorothioate nucleotides, restriction fragments, PCR products, plasmids drugs imipramine and metabolites lipids fatty acid methyl esters and triacylglycerols, silver HPLC diacylglycerols, cellular phospholipids phospholipids, diacylglycerols; silicic acid fractionation, extraction methods from cells and tissues phosphoinositols and phosphoinositides sphingomyelins and ceramides, cellular nitric oxide metabolites S-nitrosothiols peptides, proteins peptides neuropeptides size-exclusion HPLC, molecular weight estimation size-exclusion HPLC, ESI-MS aromatic amino acid containing peptides, on-line derivative spectroscopy HPLC purification for structural analysis proteins proteolysis tryptic digestion, characterization of glycopetides in reversed-phase map with HPAEC-PAD LC/MS disulfide peptides glycoproteins (see also carbohydrates) detection derivatization monosaccharides, sialic acids, amino sugar alcohols by anthranilic, o-phenylenediamine, phenylisothiocyanate reagents, respectively N- and O-glycans, pyridylamination mass spectrometry O-GlcNAc glycoproteins and glycopeptides, LC/ESI-MS LC/ESI-MS, glycosidase digestion N-linked glycans, HPAEC, MALDI-TOF refractive index detection, HPAEC; sialic acid, amino and neutral sugars PAGE (FACE) and HPLC dual analysis for oligosaccharide profiling electroblotted glycoproteins, mono- and oligosaccharides, HPAEC-PAD polyamines derivatization, dansyl chloride derivatization, benzoylated derivatives derivatization, fluorescamine, polyamines and amino acids pterins biopterins and inhibitors of biopterin biosynthesis tetrahydrobiopterin biosynthetic activities vitamins biotin panacyl bromide/crown ether fluorescence derivatization biotin and biocytin HPLC/avidin-binding assay, biotin and metabolites, 5-fmol detection limit folate metabolites retinoids thiamin and its phosphate esters vitamin B6 total pyridoxal, postcolumn derivatization various metabolites, cation-exchange HPLC 18 derivatives, free and bound forms vitamin K phylloquinone and phylloquinone 2,3-epoxide, postcolumn reduction and fluorometric detection phylloquinone, electrochemical detection phylloquinone and menaquinones, liver menaquinones; bacterial cultures, stool, intestines a

Multiple techniques covered, including HPLC.

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refs D93 D94 D95 D96 D97 D98a D99 D100 D101 D102 D103a D104 D105 D106 D107 D108 D109 D110a D111, D112 D113 D114a

D115 D116 D117 D118, D119 D120 D121-D123 D124a

D125 D126 D127 D128 D129 D130 D131a D132a D133 D134 D135 D136 D137 D138 D139 D140a D141 D142 D143 D144 D145 D146 D147 D148 D149 D150

HPLC methods), decreasing the intermethod (15-16% to 4-6%) and interlaboratory (17-19% to 5-7%) variations (D163). Vitamins. Thirty-four carotenoids, vitamin A, and two forms of vitamin E (γ- and R-tocopherol) were determined by a HPLC photodiode array detection mass spectrometric technique (D164). A reversed-phase separation of all seven vitamin B6-related compounds is reported with a separation time of less than 20 min (D165). Several ODS reversed-phase [silica and poly(vinyl alcohol) supports] and one pentafluorophenylsilica packing material were evaluated in the separation of five tocopherols (R, β, γ, δ, ζ2) and their acetyl derivatives (D166). A determination of ascorbate and dehydroascorbate in biological fluids is reported that requires no sample pretreatment, which is advantageous given the instability of these compounds to oxidation (ascorbate) and degradation in biological fluids (dehydroascorbate has a 2-min half-life in heparinized plasma) (D167). The most challenging aspect in vitamin D analysis is the determination of 1,25-dihydroxyvitamin D, which is the active form of vitamin D, but which is present at low concentrations in plasma (15-60 pg/mL). The 1,25-species is usually determined by multiple chromatographic purifications followed by a radioreceptor assay. A reversed-phase HPLC method with on-line fluorometric detection has been developed for 1,25-dihydroxyvitamin D3, providing an alternative to the radioreceptor assay (D168). However, this technique is still labor intensive, involving successive purification steps on a Bond Elut amino column and a normalphase column, collection of the fraction with subsequent fluorescence labeling (DMEQ-TAD) of the analyte, cleanup of the derivatized analyte using a Bond Elute PSA column, followed by chromatography on a normal-phase column, prior to the reversedphase HPLC analysis. The HPLC technique was compared to the radioreceptor detection technique (n ) 11) showing a 0.90 (r) correlation, with a regression line slope of 1.05 and an intercept of -2.7 (pg/mL). Lipoproteins. Lipoproteins in serum/plasma were measured by size-exclusion HPLC and by conventional methods and the results compared (D169, D170). LDL-cholesterol and HDLcholesterol values were found to be significantly higher and lower, respectively, measured by the size-exclusion HPLC method compared to conventional methods (selective precipitation of apoprotein B-containing lipoproteins for HDL-cholesterol and either the Friedewald estimation or the β-quantification for LDLcholesterol) (D169), while other work showed significantly higher and slightly lower HDL-cholesterol values determined by sizeexclusion HPLC compared to a precipitation (sodium phosphotungstate/MgCl2) and a direct method, respectively (D170). Sizeexclusion HPLC separated chylomicrons, VLDL, LDL, and HDL lipoproteins and glycerol into separate peaks detected by an online enzymatic detection of triglycerides and serum-free glycerol (D171). This method is highly sensitive and is proposed as a method for low-concentration samples such as samples from cell culture systems. Size-exclusion HPLC was also used to determine the size of LDL in a noninsulin-dependent diabetes mellitus population and compared to LDL size determinations for the same patient population by gradient gel electrophoresis, showing 0.88 (r) correlation (P < 0.001) and a high precision of measurement for the HPLC method (0.2% between-run CV) (D172).

Nitric Oxide Metabolites. Measurement of nitric oxide and its metabolites is gaining increasing importance in clinical and biomedical analysis. (a) Nitrite and Nitrate. Work reporting the determination of nitrite and nitrate in serum/blood (D173-D175); in plasma, urine, and cell cultures (D176, D177); urine (D178); and biological fluids (D179) using anion-exchange HPLC has been published. Patient groups studied for nitrite and nitrate include malaria (D173), preeclampsia (D174), and cancer (D179). Detection systems include the following: postcolumn detection involving a series of two postcolumn reactors, the first reducing nitrate to nitrite prior to detection at 540 nm after reaction with Griess reagent (30-pmol detection limit) (D176), UV detection (D174), electrochemical/ UV detection (D175), and conductometric detection (with chloride removal using a Ag+ resin) (D179). Five ion chromatography columns were tested for selectivity and resistance to overload (D174). (b) Nitrosothiols. A reversed-phase HPLC method with an on-line detection scheme involving postcolumn reaction has been developed to detect nitrosothiols at the nanomolar level (450-fmol detection limit) (D180, D181). Total nitrosothiol content in plasma has been measured down to the picomole level through a method using a reversed-phase HPLC determination of an azo dye product generated from reagents reacting with nitrosothiols (D182). (c) Other. HPLC measurement of nitric oxide synthase-related arginine derivatives has been published (D183). Glycoproteins as Markers of Disease. Measurement of different glycosylated or glycated forms of proteins is a recently emerging strategy toward identifying more specific disease markers. Glycated hemoglobin has been previously discussed. Another significant marker in this category is carbohydrate-deficient transferrin (CDT), which is a specific marker for chronically elevated alcohol consumption. In a comparison of CDT methods, a HPLC method showed better diagnostic characteristics (according to receiver-operating characteristic plots) than the established RIA technique (D184). Different isoforms of transferrin were separated by HPLC (D185, D186), which increased the accuracy of diagnosis of chronically elevated alcohol consumption (D185). Oligosaccharide profiling of the carbohydrate groups attached to the acute-phase proteins R1-proteinase inhibitor and haptoglobin by high-pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) showed characteristic patterns of glycosylation for different diseases (D187). Oligosaccharide profiling of the carbohydrate groups of R1-acid glycoprotein was done by HPAEC, with altered glycosylation patterns found in rheumatoid arthritis (D188, D189) and ulcerative colitis (D189) patients. Glycated lipoproteins (LDL and HDL) were determined by HPLC with an affinity boronate and a gel permeation column, which will be useful for studying the role of glycated lipoproteins in atherosclerosis (D190). DNA/RNA Analysis. (a) General Information. A comparative study of HPLC and CE analysis of polymerase chain reaction (PCR) products has been published (D191). A study of the effect of nucleotide sequence on the chromatography of double-stranded DNA employing a nonporous anion-exchange HPLC column was done (D192). In this work, a mixture of double-stranded DNA of constant length, with a degenerated 50-bp region, was chromatographed, showing that the chromatographic behavior of DNA was Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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governed not only by size but by nucleotide sequence; even DNA fragments having the same size and the same base content could be separated (D192). An automated HPLC system employing a 2.5-µm DEAE nonporous anion-exchange column was used for the separation of PCR products (less than 10 min) and DNA restriction fragments (D193). Reverse transcriptase-PCR combined with HPLC analysis was used to quantitatively assay for tumoral thymidylate synthase mRNA (increased amounts in tumors are predictive of resistance to treatment with 5-fluorouracil), in which DNA ranging in size from 34 to 622 base pairs eluted in 18 peaks in 22 min, (D194) and β2-adrenergic receptor mRNA, in which accurate quantification was obtained through employment of an internal standard, consisting of a competitor mRNA having the same primer sequence as the analyte but which produced a different-sized product (D195). Antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissues were determined by fast anion-exchange HPLC (nonporous) and CE, but only CE could resolve one-base differences (20mer analyte) (D196). (b) LC/MS. Several LC/ESI-MS techniques applied to nucleotide and/or oligonucleotide analysis have been published. Use of a novel additive to the mobile phase (1,1,1,3,3,3-hexafluoro-2propanol) resulted in good HPLC separation and electrospray ionization efficiency (D197, D198), demonstrating analysis of up to 75 bases for synthetic oligonucleotides, with single-base resolution achieved for oligonucleotides up to 30 bases (D197) and analysis of different phosphorylation states of nucleotides (D198). An ion-pair reversed-phase LC/ESI-MS technique has been published reporting the separation of cyclic nucleotides (D199). (c) Clinical Analytes. Denaturing HPLC is a useful technique for screening DNA samples for single-nucleotide polymorphisms and inherited mutations. Denaturing HPLC was characterized according to its ability to detect DNA sequence variation and found to have 92.5% sensitivity (n ) 40) and 100% specificity (n ) 196) (D200). Denaturing HPLC was used to identify 19 previously unreported Y chromosome polymorphisms from 718 samples (D201). Sixteen urinary ribonucleosides were separated by reversed-phase HPLC after phenylboronic acid affinity chromatography sample pretreatment, to test the speculated utility of modified (particularly methylated) nucleosides in urine (resulting from RNA degradation) in the diagnosis of malignant tumors (D202). Other Disease Markers. Methodologies currently used to measure homocysteine in plasma/serum were compared (D203). A method automating both precolumn derivatization with bromobimane and reversed-phase HPLC (with a reported chromatographic time of 6 min and a throughput of 100 samples/24 h) was used to resolve and determine important thiols in plasma and urine, including the following: homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine (D204). Cysteamine hydrochloride was proposed as an external and internal standard for HPLC assays of plasma homocysteine (D205). A study comparing the separating capabilities of reversedphase, normal, and silver ion HPLC for sterols found silver ion HPLC superior to the others with respect to separating C27 sterols differing in the number and location of the double bonds (D206). A wide range of derivatized fatty acids (C12-C54) from Mycobac318R

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teria were analyzed by LC/APCI-MS (D207). Leukotriene analysis by reversed-phase HPLC and photodiode array detection was performed with a special solid-phase extraction procedure which minimized the time of sample preparation and thus minimized inherent autoxidation leading to increased recovery, while eliminating matrix interferences (D208). Seven C18-bonded silica columns were evaluated in this leukotriene work. A LC/MS technique was developed detecting 150 different bile acids and bile alcohol conjugates (D209). A HPAEC-PAD technique for lactulose, mannitol, and glucose determination in urine for the assessment of intestinal permeability was published that has the advantage of using a simple sample preparation method compared to other techniques (D210). Fifteen quinolone antibiotics were determined by LC/ESI-MS/MS (D211), and an on-line SPE procedure for 10 cephalosporins was developed (D212). Finally, several HPLC methods for the determination of renal function were published, including the simultaneous determination of iothalamate and p-aminohippuric acid (PAH), useful for the determination of glomerular filtration rate and renal plasma flow, respectively (D213), and a HPLC method specifically determining PAH and its N-acetyl metabolite, which gave significantly lower (presumably more accurate) results in the determination of renal clearance compared to the conventional colorimetric technique (results were 35% higher for the colorimetric method) (D214). Candidate Reference Methods. HPLC has been suggested as a reference technique for serum cations (review article) (D32), sodium, potassium, calcium, and magnesium in serum (D215), creatinine in serum (D216), and selenium in biological samples (D217). A candidate reference method employing ion chromatography was compared to four routine methods in the determination of total calcium in serum (D218). A LC/APCI-MS/MS technique is proposed as a reference method for aldosterone in blood (detection limts of 10-15 pg/mL) (D219). A candidate reference method for HbA1c has been proposed (D160). SEPARATION MODES The articles given in this section are selected because of the pertinence of the properties of the separation mode (type of stationary phase, characterization or comparison of stationary phases, innovation in other separation aspects, etc.) to the chromatography of the clinical/biomedical analytes. Review articles in this topic area are also given. General Information. Features of commercially available columns, packing materials, and accessories introduced at the Pittsburgh Conference in 1997 (D220, D221) and 1998 (D222, D223) have been tabulated and summarized. Reversed Phase. A review of reversed-phase HPLC has been published (D224). Studies reporting the comparison of various commercially available reversed-phase columns according to hydrophobic and silanophilic characteristics (D225) and the characterization of nine commercially available silica-based widepore packing materials (D226) have been published. (a) Base-Deactivated Columns and Related Articles. The many aspects and effects of the silanol group in reversed-phase silica-based packing materials, including various advances pertinent to the chromatography of basic analytes, were reviewed (D227). A new column designed for basic compounds was used for the analysis of Catharanthus alkaloids (D228), while a base-

deactivated column (SCD-100) gave superior results over a standard C18 column in the analysis of urinary porphyrins (D229). A study of the type of solvent modifier (methanol, acetonitrile, THF) and the effect of the nature of the organic base (nine were studied) was performed on eight silica-based reversed-phase columns (D230). In general, acetonitrile and high-pKa bases with the least steric hindrance produced the worst peak shapes. (b) Other. Retinol and retinyl palmitate in serum were determined by C30 reversed-phase LC/APCI-MS (D231). This is advantageous over GC methodologies because a hydrolysis or derivatization step is required in the GC technique, which precludes the analysis of retinyl palmitate. A new series of packing materials was shown to be highly stable in low-pH mobile phases (even cyano and short-alkyl chain packing materials) without employing the use of bulky side groups in the reversed-phase ligand (D232). Ion Exchange. A review has been published giving example separations of synthetic (emphasized) and proteolytically derived peptides by strong cation-exchange HPLC (D233). Several papers compared the performance and characteristics of 70 lowperformance ion-exchange packing materials in protein separations and purifications (D234-D236). Chromatography of five different IgG antibodies on different ion-exchange columns (strong cationexchange, and strong and weak anion-exchange columns) was done, studying the effects of different chromatographic conditions (D237). New ion-exchange packing materials were introduced: one was the weak anion-exchanger ANX Sepharose 4 Fast Flow media (Amersham Pharmacia Biotech), offered in both low- and high-ligand coverage types, which was characterized for protein separations (D238); and the other was a highly cross-linked polymeric cation-exchange packing material from Dionex, which has a two-ligand stationary phase containing bonded carboxylate and phosphonate functional groups, being more hydrophilic than its predecessor, with the separation of amines and inorganic cations reported (D239). Finally a gradient chromatofocusing technique was developed which addresses many of the shortcomings of conventional chromatofocusing techniques (D240, D241). The technique used simple low-molecular-weight buffers (instead of the polymeric ampholytes used in the conventional technique) and employed an HPLC gradient system to readily control pH gradient slope. Separation of Chiral Compounds. A very active area of HPLC analysis of biofluids has been the separation of chiral compounds, particularly drugs. Articles cited in this review are limited to review articles and several significant articles in which multiple compounds of a particular type are determined. (a) Reviews. General reviews include reviews of various chiral stationary phases (CSPs) in various chromatographic techniques (D242), various types of commercially available CSPs in HPLC (D243), and various chiral polymer packing materials [proteins, polysaccharides and derivatives, polyamides, polymethacrylates, polyacrylamides, polymethacrylamides, polyurethanes, and synthetic polymers with chiral cavities (D244); polymethacrylates and polysaccharide derivatives (D245)]. A review of glycoprotein CSPs (R1-acid glycoprotein, cellobiohydrolase 1, ovoglycoprotein, avidin, ovotransferrin, flavoprotein, riboflavin-binding protein) has been published which covers their preparation and their chiral recognition properties and mechanisms (D246). Reviews covering chiral

separations using polysaccharide-type packing materials (D247, D248), and cyclodextrins and modified cyclodextrins [as CSPs, chiral mobile-phase additives, or chiral counterions in HPLC (D249), and as CSPs in HPLC or as mobile-phase additives in HPLC and CE (D250)] have been published. A review of chiral anion-exchange-type stationary phases based on cinchonan alkaloids for the separation of chiral acids has been published (D251). Use of sub- and supercritical fluid chromatography using CSPs has been recently reviewed (D252, D253). Column-switching techniques used in chiral separations address the problem of matrix interferences and overlapping metabolite peaks encountered in single-column chiral separation techniques, as discussed in a recent review (D254). A review covers the critical issues of chiral HPLC drug analysis, with emphasis on stability, stereoconversion, entantiomeric separation, recovery, quantification, and method validation (D255). Atomistic modeling of enantioselection applied to chiral chromatography has also been reviewed (D256). (b) Other Significant Articles. A database of 35 000 chiral separations by HPLC was developed and used to categorize the chromatographic behavior of 25 different CSPs, classifying the analytes according to 15 molecular descriptors (D257). Separation of nearly all D- and L-amino acids at the picomole level was accomplished using reversed-phase HPLC (D258). Entantiomeric separation of all phenylthiocarbamoylated amino acids was accomplished in 150 min using a tandem reversed-phase and phenylcarbamoylated/β-cyclodextrin column technique (D259). HPLC chiral separation of 21 protected amino acids (representing a cross section of reagents commonly employed in peptide synthesis) was investigated, comparing results obtained from ovomucoid (most versatile), human serum albumin, and macrocylclic antibiotic-based chiral columns (D260). Enantioselectivities of 11 steroids were determined on an amylose tris(3,5-dimethyl phenyl carbamate) column (Chiralpak AD) employing normal- and reversed-phase modes (reversed phase was better), which demonstrated higher enantioselectivities than the permethylated γand β-cyclodextrin columns (β was better) (D261). Normal-phase chiral chromatography (Chiralpak AD column) was combined online with an atmospheric pressure ionization tandem mass spectrometric detector for the analysis of several drugs and their metabolites (verapamil, oxybutynin, doxazosin, sotalol) (D262). Direct Injection HPLC. This technique refers to HPLC techniques in which samples of plasma/serum or other biofluids are directly injected onto the column without sample pretreatment to remove proteins. This is accomplished through a columnswitching design connecting a precolumn with an analytical column, through use of specialized packing materials called restricted access media (RAM), or through a combination of both. Articles referenced below are review articles or articles that report the use of RAM techniques. Although several articles reported direct injection using conventional precolumns and this technique has been shown to give superior results in comparison to direct injection techniques that solely employ RAM analytical columns (without column switching), no column-switching articles (except for those employing RAM precolumns) are cited in the present review, as little novelty was noted from previously reported techniques (D263). (a) Reviews. Review articles covering column switching and/ or RAM direct injection techniques have been published (D264Analytical Chemistry, Vol. 71, No. 12, June 15, 1999

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D266). A previous HPLC section in the Clinical Chemistry Application Review of Analytical Chemistry comprehensively reviewed this topic (D263). (b) Analytical Column RAM Techniques. Articles reporting the use of RAM analytical columns without column switching included the following: the use of a semipermeable surface column in the determination of phenylbutazone (D267) and chlorzoxazone (D268) and their metabolites in serum; the use of an internal surface reversed-phase column in the determination of a drug in plasma (D269) and dyes in the presence of DNA (D270); and the use of a column consisting of a hybrid ligand containing hydrophobic and hydrophilic phases in the determination of theophylline in serum (D271). (c) Column Switching RAM Techniques. Employment of a RAM column as a precolumn (or an extraction column) in tandem with an analytical column (using a column-switching operation) was done in the determination of cardiovascular drugs in serum (D272); in the determination of atenolol, propanolol, and ibuprofen in serum/plasma, using a RAM precolumn in which R1-acid glycoprotein is on the outer surface of the packing material and C18 ligands are in the restricted pores of the packing material (D273); and in the determination of methotrexate in plasma by ion-pairing chromatography (D274) and different drugs in various biofluids (D275) using alkyldiol-silica precolumns. Column-switching techniques employing a RAM precolumn connected to an analytical column interfaced to a mass spectrometer were published in the determination of cortisol and prednisolone in plasma and arachidonic acid in urine (using an alkyldiol precolumn) (D276) and salbutamol and clenbuterol in serum (using several types of RAM precolumns as given below) (D277). The latter technique compared three RAM precolumns, finding that the internal surface reversed-phase precolumn outperformed the semipermeable surface and the RP-18 alkyldiol silica precolumns (with respect to retention and peak width), with over 100 mL of serum being injected without noticeable deterioration of performance (D277). This precolumn lifetime is better than most of the previously reported column-switching techniques employing non-RAM precolumns [a review reported lifetimes of non-RAM precolumns to be in the range of 4-150 mL total injected plasma/ serum, with a mean of 30 mL (D263)]. Another study using a BioTrap 500 C18 RAM precolumn found less tolerance for plasma volume injected, reporting a limit of 15 mL of plasma without retention or pressure changes, while precolumn clogging and a 60% drop in efficiency of the analytical column occurred after 45 mL of plasma was injected (D278). High-Performance Affinity Chromatography (HPAC). Chromatography on red cells, biomembrane vesicles, proteoliposomes, liposomes, lipid monolayers, and plant cell walls attached to chromatographic supports has been reviewed (D279). These cell and biomolecular assembly stationary phases have been used for biomembrane affinity analyses, ion-exchange chromatography separations, membrane protein purification in detergent solutions (lipid monolayers), and group separation of macromolecules according to size and charge (plant cell walls). The use of porphyrins as ligands in the HPLC analysis of trace metals has been reviewed (D280). The employment of immunoaffinity columns coupled on-line with LC/mass spectrometry has been reviewed, discussing the analysis of plasma, urine, water, and milk 320R

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samples (D281). A column-switching technique was employed in the determination of flunitrazepam and its metabolites (nor-, 7-amino-, and 7-acetamidoflunitrazepam) in urine employing an on-line immunoaffinity extraction column (D282). This technique had extraction column lifetimes of >88 runs, total analysis times of