Journal of Chromatography B (v.824, #1-2)

This review presents recent studies on the electrospray ionisation mass spectrometry (ESI-MS) of selected N-containing drug molecules, their metabolites, formulation degradation products and process impurities taken from both studies in the author's laboratory and the recent literature using the Web of Knowledge database. Molecules of mass less than 500 Da are chosen according to selected structural classes in which they give ESI signals primarily in the positive ion mode as [M + H]+ ions. The structural classes are drugs with amine-containing side chains, drugs with N-containing saturated ring structures, drugs with N-containing unsaturated ring structures and quaternary ammonium drugs. Details are given on the fragmentations, where available, that these ionic species exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers. Fragmentation data, again where available, using electron impact mass spectrometry (EI-MS) is included for comparison. A review of applications for the period 2004–2005, again taken from the Web of Knowledge database, of the technique liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) to the detection and determination of these N-containing drug molecules in biomatrices, pharmaceutical formulations, etc., is then made. Analytical information on, for example, sample concentration techniques, LC separation conditions, recoveries from biological media, degradation products and limits of detection (LODs) are provided. Comparisons, where available, are also made with rival analytical techniques such as gas liquid chromatography–mass spectrometry (GLC–MS), capillary electrophoresis–electrospray ionisation mass spectrometry (CE–ESI-MS) and stripping voltammetry (SV).
Keywords: Electrospray ionisation mass spectrometry (ESI-MS); Fragmentation patterns; Drug analysis by liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS); Drugs of small molecular mass; Drug analysis by gas liquid chromatography–mass spectrometry (GLC–MS); Drug analysis by capillary electrophoresis–electrospray ionisation mass spectrometry (CE–ESI-MS); Stripping voltammetry (SV);

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatographyelectrospray ionization tandem mass spectrometry (LC–MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (ΔM), retention-times and full scan MS n spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6β-hydroxytropine, 6β-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25 mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.
Keywords: Anisodamine; LC–MS/MS; Metabolite;

Determination of nitrofuran metabolites in poultry muscle and eggs by liquid chromatography-tandem mass spectrometry by Jane Kelly Finzi; Jose Luiz Donato; Mauro Sucupira; Gilberto De Nucci (30-35).
The use of nitrofurans in food-producing animals has been banned in EU. Detection of the protein-bound nitrofuran metabolites is the best approach to evaluate their utilization. A fast, sensitive and reliable LC–MS–MS method is presented to analyze simultaneously the metabolites of four commonly used nitrofuran drugs, furazolidone, furaltadone, nitrofurazone and nitrofurantoin. The sample clean up was performed by a single liquid–liquid extraction step, after a hydrolysis and derivatisation process. Separation of the molecules was performed by liquid chromatography in a C18 column (100 mm × 2.1 mm, 4 μm) at room temperature. The quantitative and confirmatory determination of these metabolites was performed by multiple reactions monitoring (MRM). Limits of quantification of 0.5 ng g−1 were achieved and the total analysis was accomplished in 5 min. This protocol has been applied to identify contaminated samples of poultry muscle and egg products.
Keywords: Nitrofuran metabolites; Poultry muscle; Eggs; Mass spectrometry;

Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0–10,000 ng/mL (correlation coefficient >0.999) for sorbitol-13C6 and 250–50000 ng/mL (correlation coefficient >0.999) for fructose-13C6 in human RBCs.
Keywords: Human red blood cells; LC/APCI-MS/MS; Endogenous sorbitol and fructose; Mixed-mode solid-phase extraction; Matrix effects;

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma by S. Ramanathan; S. Karupiah; N.K. Nair; P.L. Olliaro; V. Navaratnam; W.H. Wernsdorfer; S.M. Mansor (45-50).
A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at λ ex  = 267 nm and λ em  = 443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 μg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.
Keywords: Pyronaridine; Plasma; LC; Malaria;

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists by Rosalba Gatti; Enrico Cappellin; Barbara Zecchin; Giorgia Antonelli; Paolo Spinella; Franco Mantero; Elio Franco De Palo (51-56).
A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136 ± 93 nmol/l versus 67 ± 50 nmol/l (p  < 0.001); E: 797 ± 400 nmol/l versus 408 ± 252 nmol/l (p  < 0.001)). The F/E ratio was 0.18 ± 0.11 versus 0.16 ± 0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11 ± 0.07) and cyclist individuals at rest (p  < 0.05).
Keywords: Solid phase extraction; Exercise; 11β-HSD;

A novel method was developed for the simultaneous determination of tetracycline antibiotic (TCA) residues such as oxytetracycline (OTC), tetracycline (TC), and metacycline (MTC) by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection. The procedure was based on the chemiluminescent enhancement by TCAs of the potassium permanganate–sodium sulfite–β-cyclodextrin system in a phosphoric acid medium. The separation was carried out with an isocratic elution using a mixture of acetonitrile and 0.001 M phosphoric acid. For the three TCAs, the detection limits at a signal-to-noise of 3 ranged from 0.9 to 5.0 ng/ml. The relative standard deviations for the determination of TCAs ranged from 3.1 to 7.4% within a day (n  = 11) and ranged from 2.2 to 8.6% in 3 days (n  = 9), respectively. The method was successfully applied to the determination of TCA residues in honey samples. The possible mechanism of the CL reaction was also discussed.
Keywords: Tetracycline antibiotics; HPLC; Chemiluminescence; Residues; Honey;

We describe the development and validation of analytical methodology for the determination of sphingosine 1-phosphate (S1P) in plasma. It uses solid-phase extraction (SPE) followed by an automated reversed-phase gradient HPLC column-switching system with a pre-column derivatization with o-phthalaldehyde (OPA) and fluorescence detection. The limit of quantification was determined at 100 ng/ml exogenous sphingosine 1-phosphate with a relative standard deviation for precision and accuracy <15%. The within- and between-day relative standard deviation for precision and accuracy were also less than 15%. This validated method should be suitable to quantify plasma concentration of sphingosine 1-phosphate in relatively large numbers of samples.
Keywords: Sphingosine 1-phosphate; SPE; HPLC column-switching; Automated pre-column derivatization; FLU detection; Human plasma;

A method has been developed and validated for the quantitation of midazolam, alphahydroxy-midazolam, omeprazole, and hydroxyomeprazole from one 250 μL sample of human plasma using high performance liquid chromatography coupled to tandem mass spectrometry. The method was validated for a daily working range of 0.400–100 ng/mL, with limits of detection between 2 and 15 pg/mL. The inter-assay variation was less than 15% for all analytes at four control concentrations and the samples were stable for three freeze–thaw cycles under the analysis conditions and 24 h in the post-preparative analysis matrix. This method was used to analyze samples in support of clinical studies probing the activity of the cytochrome P-450 enzyme system.
Keywords: Omeprazole; Midazolam; Analysis; LCMS; CYP2C19;

The aim of the presented study was to identify the metabolites of the new designer drug 4′-methyl-α-pyrrolidinobutyrophenone (MPBP) in rat urine using GC–MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4′-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC–MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.
Keywords: MPBP; 4′-Methyl-α-pyrrolidinobutyrophenone; Designer drug; Metabolism; Detection; GC–MS;

A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the determination of tolterodine tartrate in human plasma. With oxybutynin as internal standard, tolterodine tartrate was extracted from plasma with n-hexane: isopropanol (95:5, v/v). The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile–water (10 mM CH3COONH4, pH 3.0) = 50:50 (v/v). An aliquot of 10 μl was chromatographically analyzed on a prepacked Shimadzu Shim-pack VP-ODS C18 column (150 mm × 2.0 mm I.D.) by means of selected-ion monitoring (SIM) mode mass spectrometry. Standard curves were linear (r  = 0.9993) over the concentration range of 0.1–30.0 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection (LOD) was 0.05 ng/ml. The validated LC–ESI–MS method has been used successfully to study tolterodine tartrate pharmacokinetic, bioavailability and bioequivalence in 20 healthy male volunteers.
Keywords: Tolterodine tartrate; HPLC–ESI–MS; Human plasma; Pharmacokinetics;

An improved HPLC method for determination of carotenoids in human serum by V. Rajendran; Y.S. Pu; B.H. Chen (99-106).
An HPLC method was developed to determine the various carotenoids in human serum. A C-30 column and a mobile phase of 100% methanol (A) and 100% methylene chloride (B) with the following gradient elution were used: 90% A and 10% B in the beginning, maintained for 5 min, decreased to 78% A at 15 min, 62% A at 30 min, 52% A at 40 min, 41% A at 50 min, 38% A at 55 min, maintained for 3 min, and returned to 100% A at 65 min. A total of 21 carotenoids, including all-trans forms of lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene, as well as their 14 cis-isomers were resolved within 51 min at a flow rate of 1.0 mL/min and detection at 476 nm. all-trans-β-Carotene was found to be present in highest amount (256.3–864.2 ng/mL), followed by all-trans-lycopene (64.4–569.2 ng/mL), all-trans-lutein (137.9–450.3 ng/mL), all-trans-α-cryptoxanthin (55.7–188.2 ng/mL), all-trans-β-cryptoxanthin (43.1–134.5 ng/mL), all-trans-α-carotene (20.0–122.1 ng/mL) and all-trans-zeaxanthin (9.1–21.3 ng/mL). Similar trend was observed for cis-isomers of carotenoids.
Keywords: Carotenoids; Human serum; HPLC;

17α-Methyltestosterone (MT) is used to manipulate the gender of a variety of fish species. A high performance liquid chromatography (HPLC) internal standard method for the determination of 17α-methyltestosterone in fish feed using 3β-methoxy-17β-hydroxyandrost-5-en-7-one as internal standard (IS) has been developed. The method has been validated for the quantitation of MT in fish feed using 245 nm UV absorbance as the parent wavelength and 255 nm as a qualifier wavelength. The method was validated in the concentration range of 15.0–120 mg/kg of 17α-methyltestosterone in fish feed. Method was also found to be suitable for other feeds.
Keywords: High-performance liquid chromatography; Ultraviolet; Electrospray ionization mass spectrometry; 17α-Methyltestosterone; Fish feed; Solid phase extraction;

Increase in methylglyoxal is thought to be involved in different pathological conditions. Deamination of aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) leads to production of methylglyoxal. We have synthesized aminoacetone and developed a novel HPLC procedure for its quantitative determination. The urinary excretion of aminoacetone is approximately 20–30 μg/mouse/day, and the concentration is about 0.5 μg/g in mouse liver and small intestine. SSAO inhibitor increases aminoacetone levels in both tissues and urines. Results confirm that aminoacetone is an endogenous substrate for SSAO. However, data also indicate that deamination is not the only catabolic pathway for aminoacetone.
Keywords: Aminoacetone; SSAO; Methylglyoxal; HPLC;

4-Methylthioamphetamine (4-MTA) is a scheduled designer drug that has appeared on the illicit drug market and led to several non-fatal or even fatal poisonings. Only few data are available on its metabolism. The first aim of this study was to identify the 4-MTA metabolites in human urine and then to study whether the authors’ STA procedure is suitable for screening for and identification of 4-MTA and/or its metabolites in urine. After enzymatic cleavage of conjugates, solid-phase extraction (SPE) and acetylation the following metabolites could be identified by full-scan gas chromatography–mass spectrometry (GC–MS): deamino-oxo 4-MTA, deamino-hydroxy 4-MTA, ring hydroxy and β-hydroxy 4-MTA. 4-MTA sulfoxide could be identified as possible artifact. In urine samples after enzymatic hydrolysis, acidic extraction, and methylation, 4-methylthiobenzoic acid could be identified. The authors’ systematical toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis, liquid–liquid extraction (LLE) and acetylation allowed detection of 4-MTA as target analyte plus all the above-mentioned metabolites with the exception of 4-methylthiobenzoic acid. The extraction efficiency of 4-MTA was approximately 70% and the limit of detection (LOD) was 30 ng/ml (S/N 3).
Keywords: 4-Methylthioamphetamine; 4-MTA; Designer drug; Metabolism; GC–MS;

Vitamin C plays a central role in the body. One of its important functions is its role as an antioxidant, and accurate measurements are important for interpretations of this role. However, its reactive nature and instability complicates the assessment, especially in biological samples. A high-throughput chromatographic method using monolithic column and UV-detection was developed for the assessment of plasma ascorbic acid and total ascorbic acid. The method showed excellent analytical sensitivity, specificity, precision, recovery and linearity during the validation study. The method was used for the assessment of ascorbic acid and total ascorbic acid during several clinical studies.
Keywords: Vitamin C; Ascorbic acid; Dehydroascorbic acid; Plasma; Monolithic column;

Use of graphitised carbon negative ion LC–MS to analyse enzymatically digested glycosaminoglycans by Niclas G. Karlsson; Benjamin L. Schulz; Nicolle H. Packer; John M. Whitelock (139-147).
Capillary liquid chromatography–mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M − H] ions using a standard electrospray interface and flow rate between 6–10 μL/min. Graphitised carbon liquid chromatography–fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M − H] ions (low sulphated disaccharides) or of the [M + Na − 2H] ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.
Keywords: Proteoglycans; Hyaluronic acid; Keratan sulphate; Heparin; Heparan sulphate;

An LC–MS–MS method for the determination of cyclizine in human serum by A. Mohammadi; I. Kanfer; V. Sewram; R.B. Walker (148-152).
Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid–liquid extraction of 200 μl of serum with dichloromethane after the addition of 100 μl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna® C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5–100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.
Keywords: LC–MS–MS; Cyclizine; Chlorcyclizine; Human serum;

Ftorafur (FT), an oral prodrug of 5-FU, is part of UFT and S1, two oral prodrugs widely used in digestive tract cancer. We set up a liquid chromatography tandem mass spectrometry (LC/MS–MS) method, chosen for its specificity of detection, for simultaneously measuring in human plasma FT, 5-FU and 5-FUH2. Separation was performed on a Hypercarb column. Linearity, precision and accuracy were validated in the concentration range studied for each compound. This simple and reliable LC/MS–MS method allows specific, sensitive and reproducible quantification of FT, 5-FU and FUH2 in human plasma and can be applied to further pharmacokinetic studies in patients treated with FT-based prodrugs.
Keywords: Tégafur; UFT; S1; 5-Fluorouracil; 5-Fluorodihydrouracil; LC/MS–MS;

In order to determine the collagen content of small amounts of skin tissue, we developed a new, simple and highly sensitive method of measuring the quantity of hydroxyproline (Hyp) using isocratic high performance liquid chromatography (HPLC) with a fluorogenic agent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F). The recovery rate of Hyp and reproducibility of the assay were high, and the test was sensitive enough to detect Hyp in less than 1 mg of skin tissue. This method is clinically useful for ensuring accurate diagnosis and for monitoring specific skin conditions using small human skin samples collected in biopsies.
Keywords: Hydroxyproline; Collagen; NBD-F; Skin; High performance liquid chromatography (HPLC);

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-μL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0–220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.
Keywords: Benzo[a]pyrene; Benzo[a]pyrene-dione; Cytochrome P450; Post-column; Zinc reduction; High-performance liquid chromatography (HPLC); Metabolites;

A very simple and direct method for determination of uric acid, in various biological matrices, based on high-performance liquid chromatography and mass spectrometry is described. Chromatographic separations were performed with a stationary phase Zorbax Sax Column, an anion exchange resin, with 50% sodium citrate 1 mM at pH 6.5 and 50% acetonitrile as mobile phase delivered at a flow rate of 1 ml/min. The detector counted negative ions by monitoring m/z 167.1, which corresponds to the urate anion. The method does not use an internal standard but quality control samples were used. Intra-day precision ranged between 1.1 and 1.5%, whereas inter-day precision was between 1.3 and 2.8% (n  = 5) working with some selected standards. Recovery tests of added standard have been successfully performed in urine and saliva samples, thus showing an appropriate accuracy of the method. The limit of quantitation found was 70 μg/l. Different urine and saliva samples were analyzed using an alternative analytical methodology based on an enzymatic reaction and photometric detection at 520 nm, resulting both methods comparable at a 95% confidence level. The method has been also applied to the determination of trace amounts of uric acid in the core of some selected calcium oxalate renal calculi.
Keywords: Uric acid; Liquid chromatography; Mass spectrometry; Renal calculi;

A LC–tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 μm, 100 mm × 2.0 mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mL min−1. The injection volume for each sample was 20 μL with a total run time of 7.5 min. This method was validated in the range 25–4000 nM (r 2  = 0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.
Keywords: BGC945; BGC638; Validation; Liquid chromatography–tandem mass spectrometry;

Optimization of protein identification from digests as analyzed by capillary isoelectric focusing-mass spectrometry by Henricus F. Storms; Robert van der Heijden; Ubbo R. Tjaden; Jan van der Greef (189-200).
Capillary isoelectric focusing (CIEF) is a high-resolution separation technique for the analysis of peptides and protein digests. When coupled to ion trap-mass spectrometry (CIEF-MS) the unique separation mechanism is combined with a highly efficient detection system. In an earlier report, we described aspects of separation and interfacing in connection to the analysis of a digest of set of standard proteins. Now, we report on different aspects of the process of protein identification. Sequest software parameters were optimized by using a standard protein digest. These settings were used for the analysis of periplasmic proteins from Escherichia coli. Since in CIEF peptides are focused according to their pI values, the mobilization time of a particular peptide is dependent on its pI value. Based on this relation, the identification of some peptides was facilitated. Furthermore, the Sequest settings that were used could be evaluated. In total, 159 proteins were identified in a single run.
Keywords: Capillary isoelectric focusing; Mass spectrometry; Protein analysis; Data analysis; Sequest;

Estimation of pK a values using microchip capillary electrophoresis and indirect fluorescence detection by Christa A. Currie; William R. Heineman; H. Brian Halsall; Carl J. Seliskar; Patrick A. Limbach; Francisco Arias; Kenneth R. Wehmeyer (201-205).
Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK a values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK a values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK a values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK a values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.
Keywords: Microchip capillary electrophoresis; pK a; Indirect fluorescence detection;

Tetrahydrocurcumin in plasma and urine: Quantitation by high performance liquid chromatography by Dennis D. Heath; Milagros A. Pruitt; Dean E. Brenner; Aynun N. Begum; Sally A. Frautschy; Cheryl L. Rock (206-212).
Tetrahydrocurcumin (THC), one of the major metabolites of curcumin, exhibits many of the same physiologic and pharmacological activities as curcumin and in some systems may exert greater antioxidant activity than curcumin. However, evaluation of clinical efficacy is limited by lack of sensitive methods for quantifying intake/absorption in blood or urine. We have developed a sensitive high performance liquid chromatography (HPLC) analytical method for detection of THC in plasma and urine. The method involves extracting the THC from 0.2 mL samples with 95% ethyl acetate/5% methanol, and β-17-estradiol acetate as an internal standard. Analysis with a reversed-phase C18 column and UV detection at 280 nm demonstrates linear performance from 0.050 to 6.0 μg/mL in plasma, and 0.060 to 6.0 μg/mL in urine. The coefficients of variation for intra- and inter-assays were each <8.6%. The average recovery of THC from plasma and urine was greater than 98.5%. These data demonstrate a rapid, sensitive and accurate method for HPLC quantification of THC in plasma and urine.
Keywords: Curcumin; Tetrahydrocurcumin; HPLC; Plasma; Urine;

For a pharmacokinetic–pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC–MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 °C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5–500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (≤20% at lower limit of quantification (LLQ) and ≤15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.
Keywords: Heroin; Acetylcodeine; Cocaine; Methadone; LC–MS–MS;

Reaction between iodine and azide ion induced by mercaptopyridines and mercaptopyrimidines was utilized as a detection system in TLC and HPTLC. The developed plates were sprayed with a freshly prepared mixtures of sodium azide and starch solution adjusted to pH 5.5, and exposed to iodine vapour. The spots became visible as white spots on violet-grey background. The iodine–azide detection system has been proved to be the most favourable and enabled to detect quantities per spot in the range of 1–20 pmol (HPTLC) and 1–60 pmol (TLC). The iodine–azide tests were compared with other visualizing techniques commonly used in planar chromatography (iodine vapour and UV254). The developed method was applied to detection of thiopental in biological samples.
Keywords: Iodine–azide reaction; Mercaptopyridines; Mercaptopyrimidines; TLC; HPTLC;

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and possible human carcinogen present in diesel exhaust and airborne particulate matter. Nitroreduction is believed to play a crucial role in nitroarene activation and mutagenicity; however, quantification of nitroreduction rate in mammalian samples has proved difficult. In this study, we present a sensitive method to quantify 3-nitrobenzanthrone reductase activity in murine tissues via normal-phase HPLC with fluorescence detection of the reduced product 3-aminobenzanthrone (3-ABA). Calibration linearity was obtained for pure 3-ABA concentrations of 1–500 ng/ml (r 2  > 0.99), with a detection limit of 0.25 ng/ml (S/N = 3). Incubation time, substrate concentration, and protein concentration in the reaction mixture were optimized, and the detection limit of the enzyme assay is 0.97 pmol/min/mg protein. The apparent K m and V max for post-mitochondrial supernatant from Muta™Mouse liver (i.e., liver S9) were 23.9 μM and 70.2 pmol/min/mg protein, respectively. Analysis of replicate samples of Muta™Mouse liver and lung S9 yielded mean activity values of 39.0 ± 3.0 and 61.1 ± 4.3 pmol/min/mg, respectively. ANOVA revealed significant effects of tissue type and incubation condition (i.e., with or without N2). The results show significantly higher activity in lung, and, in contrast to that observed for 1-nitropyrene, incubation in open air (i.e., without N2 bubbling) causes only a marginal decrease in activity. Quantification of 3-NBA nitroreductase activity in murine tissues will provide insight into the published tissue-specific mutagenic activity of 3-NBA.
Keywords: Nitroreductase; Nitroarenes; HPLC; 3-Aminobenzanthrone;

A new sensitive column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the simultaneous determination of rabeprazole, a proton pump inhibitor, and its active metabolite, rabeprazole thioether in human plasma. Rabeprazole, its thioether metabolite and 5-methyl-2-[{4-(3-methoxypropoxy)-3-methyl pyridin-2-yl} methyl sulfinyl]-1H-benzimidazole, as an internal standard were extracted from 1 ml of plasma using diethyl ether–dichloromethane (9:1, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 35 mm × 4.6 mm I.D.) for clean-up and column II (C18 Grand ODS-80TM TS analytical column, 5 μm, 250 mm × 4.6 mm I.D.) for separation. The peak was detected with an ultraviolet detector set at a wavelength of 288 nm, and the total time for chromatographic separation was ∼25 min. Mean absolute recoveries were 78.0 and 88.3% for rabeprazole and rabeprazole thioether, respectively. Intra- and inter-day coefficient variations were less than 6.5 and 4.5% for rabeprazole, 3.6 and 5.3% for rabeprazole thioether, respectively, at the different concentration ranges. The validated concentration ranges of this method were 1–1000 ng/ml for rabeprazole and 3–500 ng/ml for rabeprazole thioether. The limits of quantification were 1 ng/ml for rabeprazole and 3 ng/ml for rabeprazole thioether. The method was suitable for therapeutic drug monitoring and was applied to pharmacokinetic study in human volunteers.
Keywords: Rabeprazole; Rabeprazole thioether; HPLC;

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method (LC–MS–MS) had been developed and validated for the quantitation of astragaloside IV (AGS-IV)-an active constituent of Radix Astragali in rat plasma. Assay method was developed by a series of operations described as below. The plasma proteins were precipitated with acetonitrile and digoxin was used as the internal standard (I.S.). The sample solution containing astragaloside IV and the I.S. were obtained and subsequently injected into a LC–MS–MS system following by a gradient elution at a slow flow rate combined with a valve diversion during the liquid chromatography. Chromatographic separation was achieved on a C4 (2.1 mm × 10 mm) column with a gradient mobile phase comprised of 90% methanol in water and 10 mM ammonium acetate buffer. The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction-monitoring (MRM) modes were m/z 785.5 (precursor ion) to m/z 143.2 (product ion) for AGS-IV and m/z 781.2 (precursor ion) to m/z 243.3 (product ion) for digoxin (I.S.). The method was validated over a linear range of 1–1000 ng/ml. The low limit of quantitation was 1.0 ng/ml. Results from a 3-day validation study demonstrated that the developed method possessed good precision (CV% values were between 5.9 and 7.6%) and accuracy (96.5–102.1%) across the calibration range. The recoveries were 91 and 90% for astragaloside IV and I.S., and no significant matrix effects were observed. QC samples were stable when kept at room temperature for 4 h, at −20 °C for 4 weeks, and after three freeze/thaw cycles.
Keywords: Astragalus membranaceous; Astragaloside IV;

Quantification of lipoic acid in plasma by high-performance liquid chromatography–electrospray ionization mass spectrometry by Jun Chen; Wenming Jiang; Jia Cai; Weixing Tao; Xiaoling Gao; Xinguo Jiang (249-257).
A sensitive and specific liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS) method has been developed and validated for the identification and quantification of lipoic acid (LA) in human plasma. LA and the internal standard, naproxen, were extracted from a 500 μl plasma sample by one-step deproteination using acetonitrile. Chromatographic separation was performed on a Zorbax SB-C18 Column (100 mm × 3.0 mm i.d. with 3.5 μm particle size) with the mobile phase consisting of acetonitrile and 0.1% acetic acid (pH 4, adjusted with ammonia solution) (65:35, v/v), and the flow rate was set at 0.3 ml/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method was linear over the concentration range of 5–10,000 ng/ml for LA. The intra- and inter-day precisions were less than 7% and accuracy ranged from −7.87 to 9.74% at the LA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around time. The method has been successfully applied to a clinical pharmacokinetic study of LA in 10 healthy subjects.
Keywords: Lipoic acid; HPLC–ESI-MS;

A quantitative method for measuring testosterone (T) concentrations in rat plasma was developed using ethyl oxime and acetyl ester derivatization and liquid chromatography–atmosphere pressure chemical ionization tandem mass spectrometry (LC–APCI-MS/MS). The method utilizes a solid phase extraction with Varian Bond Elut C18, a derivatization process to form testosterone ethoxime acetate and LC–APCI-MS/MS with a reversed phase LC and a C8 column. This method is capable of detecting testosterone concentrations as low as 0.2 ng/ml in a 0.05 ml sample of rat plasma. This method can be used as a sensitive chromatography-based assay for small sample volumes of rat blood.
Keywords: Testosterone; Liquid chromatography–atmosphere pressure chemical ionization mass spectrometry; Derivatization; Acetate; Oxime;

Quantification of rotavirus-like particles by gel permeation chromatography by Jimmy A. Mena; Octavio T. Ramírez; Laura A. Palomares (267-276).
There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.
Keywords: Rotavirus-like particles; HPLC; Gel permeation;

A rapid, sensitive, and reproducible method was developed for quantitative determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and its biodegradation intermediates, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. RDX, MNX, DNX, or TNX was extracted from soil by pressurized liquid extraction (PLE), followed by cleanup using florisil. Instrumental analysis was performed using gas chromatography with electron capture detection (GC–ECD), which was highly sensitive to the parent explosive and its metabolites. The method detection limits (MDLs) were 0.243, 0.095, 0.138, and 0.057 ng/g for RDX, MNX, DNX, and TNX, respectively. The method gave high recovery (98–102%), good precision (0.22–5.14%), and reproducibility, and proved to be suitable for real world sample analysis.
Keywords: Explosives; RDX; Gas chromatography (GC); Electron capture detection (ECD); Soil analysis; Pressurized liquid extraction (PLE);

We developed a LC–MS/MS method for the quantitative determination of the mercapturic acid (MA) metabolites of acrylamide (AA) AAMA and of its oxidative metabolite glycidamide (GA) GAMA in urine samples from the general population. The method requires 4 mL of urine which is solid phase extracted prior to LC–MS/MS analysis. The metabolites are detected by ESI-tandem mass spectrometry in negative ionisation mode and quantified by isotope dilution. Detection limits ranged down to 1.5 μg/L urine for both AAMA and GAMA. The imprecision expressed as R.S.D. lay between 2% and 6% for both analytes (intra- and inter-assay). First results on a small group of 29 persons out of the general population ranged from 5 to 338 μg/L AAMA and <LOD to 45 μg/L GAMA in urine. Only in one urine sample GAMA could not be detected. With this sensitive, reliable and rapid method we can determine the internal exposure of the general population to acrylamide in terms of the mercapturic acids. Especially the determination of GAMA is of great toxicological importance because GA is the ultimate carcinogenic agent in AA metabolism. The method therefore provides better insight into the metabolism of acrylamide in humans and furthermore supports risk assessments.
Keywords: Acrylamide (AA); Glycidamide (GA); Biological monitoring; Mercapturic acids; Metabolites; N-Acetyl-S-(2-carbamoylethyl)-l-cysteine (AAMA); N-(R/S)-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-l-cysteine (GAMA);

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).
Keywords: Pyridylamination of glucans with terminal hemiacetal; SEC-mass/molar; Absolute molecular weight distribution: Mass fractions; Absolute molecular weight distribution; Molar fractions; Narrow distributed non-branched AP-glucans; Broad distributed branched AP-glucans; Mixed type branched AP-glucans;

Ng-hydroxy-l-arginine has been described as an intermediate in the nitric oxide pathway. Its electrochemical oxidation was studied by capillary electrophoresis using either electrospray ionisation mass spectrometry or diode array detection. Three stable end products and unstable intermediates were found using these techniques. These results permitted to propose an electrochemical oxidation scheme of this molecule.
Keywords: Ng-hydroxy-arginine; Oxidation; Capillary electrophoresis;

Orthologous proteomes, universal protein networks conserved from bacteria to mammals, dictate the core functions of cells. To isolate mammalian protein sequences that interact with bacterial signaling proteins, a BLASTP genome search was performed using catalytic domains of bacterial phosphoryl-transfer enzymes as probes. A [32P]phosphoryl-transfer assay of these mammalian cDNA-expressing Escherichia coli cells was used to screen proteins retrieved from the database. Here we report that the expression of a human protein, named calphoglin, resulted in a significant increase in the phosphorylation of a 55-kDa protein in E. coli. The phosphorylation of the 55-kDa protein was acid-stable and its isoelectric point was determined to be 5.4. The 55-kDa protein was sequentially purified from an E. coli extract using three chromatography and two-dimensional polyacrylamide gel electrophoresis. Finally, the 55-kDa protein was purified 830-fold to homogeneity and the N-terminal amino acid sequence was analyzed. The sequence obtained, AIHNRAGQPAQQ, was identical to the N-terminal amino acids of E. coli phosphoglucomutase (PGM). This method may be applicable to the detection and analysis of other orthologous proteomes.
Keywords: Calphoglin; Phosphoglucomutase; Protein phosphorylation; Genome; Orthologue proteome;

Determination of metformin in human plasma by high-performance liquid chromatography by Hossein Amini; Abolhassan Ahmadiani; Parisa Gazerani (319-322).
A simple, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of antihyperglycemic agent metformin in human plasma using a novel sample extraction procedure. Liquid–liquid extraction of metformin and ranitidine (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (50:50, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a silica column (250 mm × 4.6 mm, 5 μm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (25:75, v/v), pH 6. The limit of quantification (LOQ) was 15.6 ng/ml and the calibration curves were linear up to 2000 ng/ml. The mean absolute recoveries for metformin and internal standard using the present extraction procedure were 98 and 95%, respectively. The intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 8.3%.
Keywords: Metformin; Liquid–liquid extraction;

An oligomerized β-cyclodextrin ligand coupled to brominated allyl-group substituted Sepharose HP has been used for the one-step purification of polyphenolic epigallocatechin gallate (EGCG), an important antioxidant, by isocratic hydrogen bond adsorption chromatography. With a sample load of 1.33 mg crude green tea polyphenolic extract per ml column packing and with water/ethanol/acetonitrile (57/30/13, v/v) as the optimum mobile phase, an EGCG purity of about 98% with a recovery of approximate 73% could be achieved by proper peak cutting. After about 10 sample applications, the column performance started to deteriorate but could be regenerated to its original function by cleaning with 0.35 M NaOH.
Keywords: β-Cyclodextrin; Tea polyphenols; EGCG; Catechin;

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak® CN HP column with an acetonitrile–aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (λ  = 350 nm) from the postcolumn iodine–azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N = 3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N = 3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.
Keywords: Iodine–azide reaction; Thiopental; HPLC;

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection by Simon N. Muchohi; Kenneth Obiero; Gilbert O. Kokwaro; Bernhards R. Ogutu; Isaiah M. Githiga; Geoffrey Edwards; Charles R.J.C. Newton (333-340).
A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid–liquid extraction with a mixture of n-hexane–dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi ® Max RP analytical column (150 mm × 4.6 mm i.d.; 4 μm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)–acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r 2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1 ± 5.5% (n  = 6) and 72.4 ± 5.9% (n  = 7), respectively; for OZP at 200 ng the value was 68.2 ± 6.8% (n  = 14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n  = 7 in all cases) and 6.6% (n  = 8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n  = 7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of ±20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.
Keywords: Lorazepam; High-performance liquid chromatography; Analysis in plasma; Pharmacokinetics; Children;

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 °C. With the mobile phases consisting of acetonitrile–water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 μl, the column temperature 40 °C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5–150.0 μg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from −0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.
Keywords: Derivatization, LC; Detection, LC; Valienamine; p-Nitrofluorobenzene; Validamycin A;

Author Index (351-353).

Keyword Index (354-360).