Journal of Chromatography B (v.810, #1)

News Section (N1-N2).

Evaluation of DNA adduction of AZT in peripheral blood leukocytes of HIV-infected individuals by 32P-post-labeling thin-layer chromatography: a feasibility study by Jerry M. Huang; Kathryn Anastos; Esther Robison; Ruijin Shi; Kathy Freeman; Howard Strickler; J.J. Steinberg (1-6).
3′-Azido-3′-deoxythymidine (AZT, Zidovudine) has been effectively used for HIV infection treatment. It inhibits virus reproduction through viral reverse transcriptase inhibition. However, the side effects of this anti-retroviral drug might be cumulative, particularly in its effects on the patients’ DNA. As a nucleoside analogue, AZT might incorporate into hosts’ DNA, and then form DNA adducts. This may result in potential long-term risks of mutagenesis in AIDS patients who received therapy. In this feasibility study, a 32P-post-labeling thin-layer chromatography (TLC) assay is successfully used to measure AZT–DNA analogue and adducts formed in peripheral blood leukocytes of AZT treated patients. There are DNA analogue/adducts measured in all four AZT treated patients’ DNA specimens. This assay is reliable with the significant coefficient of correlation in both intra-assay (r = 0.8761, P = 0.0001) and inter-assay (r = 0.8761, P = 0.0001).
Keywords: DNA; 3′-Azido-3′-deoxythymidine;

5-[2,4-Dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl]methyl]benzamide (I, MK-0767 or KRP-297, Fig. 1), is a dual α/γ peroxisome proliferator-activated receptor (PPAR) agonist. A LC–MS/MS method for the determination of I in human plasma has been successfully developed, validated and applied to clinical programs. The analyte and internal standard (II) are extracted from 0.05 mL plasma via solid phase extraction (SPE). HPLC is used for the separation of I and II from possible co-extracted endogenous and other compounds. Detection is by MS/MS in multiple reaction monitoring (MRM) mode using a TurboIonSpray® probe. The whole sample preparation is automated by using a Packard Multiprobe liquid handling system. The linear range is 4–2000 ng/mL in plasma. Recoveries were 71.1% and 69.4% for I and II, respectively. The method exhibited good linearity, reproducibility and sensitivity, selectivity and robustness when used for the analysis of clinical samples.
Keywords: MK-0767; Peroxisome proliferator-activated receptor;

Monolithic peptidyl sorbents for comparison of affinity properties of plasminogen activators by Evgenia G. Vlakh; Alexander Tappe; Cornelia Kasper; Tatiana B. Tennikova (15-23).
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
Keywords: Fibrinolysis; Tissue plasminogen activator; Streptokinase; Pro-urokinase; Macroporous disks; Solid phase peptide synthesis; GMA–EDMA monoliths; Ligand immobilization; Short monolithic columns;

Determination of the new HIV-protease inhibitor atazanavir by liquid chromatography after solid-phase extraction by S. Colombo; N. Guignard; C. Marzolini; A. Telenti; J. Biollaz; L.A. Decosterd (25-34).
An HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz is proposed here for the simultaneous analysis of the new HIV protease inhibitor atazanavir (ATV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection. After viral inactivation by heat (60 °C for 60 min), plasma (600 μl) with clozapine (internal standard) is diluted 1 + 1 with phosphate buffer pH 7 and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with 2 × 500 μl of a solution of 0.1% H3PO4 neutralised with NaOH to pH 7. ATV is eluted with 3 × 500 μl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 μl MeOH/H2O 50/50. A 40 μl volume is injected onto a Nucleosil 100–5 μm C18 AB column. ATV is analysed by UV detection at 201 nm using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.14. The mobile phase also contains 0.02% sodium heptanesulfonate, enabling an excellent separation of ATV from the other HIV protease inhibitors (PIs) amprenavir, indinavir, saquinavir, ritonavir, lopinavir, nelfinavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. The calibration curves are linear up to 10 μg/ml, with a lower limit of quantification of 0.2 μg/ml. The mean absolute recovery of ATV is 96.4 ± 3.2%. The method is precise with mean inter-day CVs within 1.1–6.1%, and accurate (range of inter-day deviations +0.3 to +2.3%). The method has been validated and is currently applied to the monitoring of ATV in HIV patients.
Keywords: Atazanavir; Protease inhibitors; Non-nucleoside reverse transcriptase inhibitors;

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of halofuginone in plasma by Robert A. Parise; Barney R. Sparrow; John W. Merrill; Irma M. Grossi; Joseph M. Covey; James O. Peggins; Merrill J. Egorin (35-40).
An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 μ Polar RP 80A (4 μm, 100 mm × 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1 mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.
Keywords: Halofuginone;

Detection of cariogenic bacterial genes by microchip electrophoresis by Koji Karasawa; Hidetoshi Arakawa; Takeshi Igarashi; Nobuichi Goto; Masako Maeda (41-47).
Allele-specific PCR primers were designed, based on the dextranase (dex) gene, to identify Streptococcus mutans and Streptococcus sobrinus in dental plaque; subsequently, PCR products were detected via microchip electrophoresis (ME). In order to amplify the dex gene fragment of S. mutans and S. sobrinus, the following two PCR methods were established. Duplex allele-specific PCR primers were designed on a region of low DNA homology; furthermore, 211 and 126-bp fragments were amplified for S. mutans and S. sobrinus, respectively. Common PCR primer for single allele-specific PCR was designed so as to sandwich a region exhibiting high homology and amplify PCR product of different DNA size due to deletion of small DNA fragment in two dex genes. S. mutans and S. sobrinus were amplified, leading to the generation of 202 and 226-bp products, respectively. Analysis of DNA base size by ME in order to achieve efficient separation employed a polymer mixture consisting of hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO). In the presence of a polymer mixture of 0.125% PEO/0.6% HPMC, two PCR products were obtained, displaying degree of separation of 226 bp/202 bp of 2.67 (Rs). Reproducibility (CV%, n = 7) was 0.3%; additionally, separation time was approximately 85 s. This method was applied to the detection of S. mutans and S. sobrinus in dental plaque. Detection of the dex genes of S. mutans and S. sobrinus characterized by quickness, precision and high sensitivity was possible.
Keywords: Microchip electrophoresis; Dextranase gene; Streptococcus mutans; Dental caries;

We report a method for determining fluorinated organic compounds such as perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA) and perfluorooctanesulfonylamide (PFOSA) in human blood samples by column-switching liquid chromatography–electrospray mass spectrometry. The sample preparation prior to solid phase extraction (Waters Oasis HLB extraction column) involved simply mixing plasma sample with internal standard followed by centrifugation and extraction. The compounds were separated by reversed-phase chromatography with a C8 column, and detected by mass spectrometry using selected ion monitoring in the negative mode. The average recoveries of PFOS, PFOA and PFOSA ranged from 82.2 to 98.7% (R.S.D.: from 2.0 to 5.2%, n = 6). The limits of quantitation of PFOS, PFOA and PFOSA at signal to noise (S/N = 10) were 0.5, 0.5 and 1.0 ng ml−1. The method enables the precise determination of standards and can be applied to the detection of PFOS, PFOA and PFOSA in human plasma samples for monitoring human exposure.
Keywords: Perfluorooctane sulfonate; Perfluorooctanoate; Perfluorooctanesulfonylamide;

We describe a rapid GC/MS assay for amphetamine-type stimulant drugs (ATSs) and structurally related common medicaments in blood, serum, oral fluid and urine samples. The drugs were extracted from their matrices and derivatized with heptafluorobutyric anhydride (HFBA) in a single step, using the following procedure: 100 μl (oral fluid) or 200 μl (blood, serum, urine) of the sample were mixed with 50 μl of alkaline buffer and 500 μl of extraction–derivatization reagent (toluene + HFBA + internal standard), centrifuged, and injected into a GC/MS apparatus. As revealed by the validation data this procedure, with its limit of quantitation being set at 20 ng/ml for oral fluid, 25 ng/ml for blood or 200 ng/ml for urine, is suitable for screening, identification and quantitative determination of the ATSs and related drugs in all the matrices examined. Thus, time-consuming and expensive multiple analyses are not needed, unless specifically required.
Keywords: Amphetamine-type stimulant drugs;

A rugged, reproducible, multi-dimensional LC–MS system was developed to identify and characterize proteins involved in protein–protein interactions and/or protein complexes. Our objective was to optimize chromatographic parameters for complex protein mixture analyses using automated peptide sequence recognition as an analytical end-point. The chromatographic system uses orthogonal separation mechanisms by employing strong cation exchange (SCX) in the first dimension and reversed phase (RP) in the second dimension. The system is fully automated and sufficiently robust to handle direct injections of protein digests. This system incorporates a streamlined post analysis results comparison, called DBParser, which permitted comprehensive evaluation of sample loading and chromatographic conditions to optimize the performance and reproducibility. Peptides obtained from trypsin digestion of a yeast soluble extract provided an open-ended model system containing a wide variety and dynamic range of components. Conditions are described that resulted in an average (n = 4) of 1489 unique peptide identifications, corresponding to 459 non-redundant protein sequence database records (SDRs) in the 20 μg soluble fraction digest.
Keywords: Multi-dimensional LC–MS; 2D-LC–MS/MS; Peptide mixtures; Sequence database records (SDRs); S. cerevisiae; DBParser;

Simultaneous determination of levofloxacin, gatifloxacin and moxifloxacin in serum by liquid chromatography with column switching by Hoang Anh Nguyen; Jean Grellet; Boubakar B. Ba; Claudine Quentin; Marie-Claude Saux (77-83).
Liquid chromatography with a column-switching technique was developed for simultaneous direct quantification of levofloxacin, gatifloxacin and moxifloxacin in human serum. Serum samples were injected on a LiChroCART® 4-4 pre-column (PC) filled with a LiChrospher® 100 RP-18, 5 μm where fluoroquinolones (FQs) were purified and concentrated. The FQs were back-flushed from the PC and then separated on a Supelcosil ABZ+ Plus (150 mm × 4.6 mm i.d.) analytical column with a mobile phase containing 10 mM phosphate buffer (pH 2.5), acetonitrile (88:12, v/v) and 2 mM tetrabutyl ammonium bromide. The effects of ion-pair reagents, buffer type, pH and acetonitrile concentrations in the mobile phase on the separation of the three FQs were investigated. Fluorescence detection provided sufficient sensitivity to achieve a quantification limit of 125 ng/ml for levofloxacin and moxifloxacin; 162.5 ng/ml for gatifloxacin with a 5 μl sample size. The on-line process of extraction avoids time-consuming treatment of the samples before injection and run time is shortened. The recovery, selectivity, linearity, precision and accuracy of the method are convenient for pharmacokinetic studies or routine assays.
Keywords: Column switching; Levofloxacin; Moxifloxacin; Gatifloxacin;

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography–mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC18 narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3–10 μg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of −6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC–ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.
Keywords: Edelfosine; Drug delivery systems; Antitumoral agents;

Studies of the antioxidant defense system and the related metabolic pathways are often complicated by cumbersome analytical methods, which require separate and multi-step extraction and chemical reaction procedures. Further, assaying multiple parameters is limited because of the usual small sample amounts. High-performance liquid chromatography coupled with a coulometric multi-electrode array system provides us high specificity and sensitivity to measure electrochemically oxidizable compounds in biological samples. In contrast to previously reported methods with two columns in series and a complex gradient elution profile, we have developed an automated procedure to simultaneously measure multiple redox-active low-molecular weight compounds that utilizes a single column with a simplified binary gradient profile. No other chemical reactions are necessary. In order to reduce the running time and yet achieve a reproducible retention time by the auto sampler injection, our gradient elution profile was modified to produce a shorter equilibration time, stable retention time, and a reduced cost per test.
Keywords: Coulometric multi-electrode array system; Redox-active compounds;

A specific capillary electrophoresis–time-of-flight mass spectrometry (CE–TOFMS) method for the determination of serotonin (5HT) and its precursors tryptophan (Trp) and 5-hydroxytryptophan (5HTP) in human platelet rich plasma is described. The analytes were removed from the plasma samples and preconcentrated by solid phase extraction (SPE) on mixed mode cation-exchange sorbents. The SPE recoveries were 71.6 ± 3.1 for 5HT, 91.0 ± 2.8 for Trp, and 95.3 ± 5.9% for 5HTP. Deuterated analogues of 5HT and Trp were used as internal standards for quantitation purposes. Submicromolar detection limits were obtained for standard mixtures of all compounds and their deuterated isotopes, except 5HTP, which had detection limits in the low micromolar range. The potential usefulness of this method in the clinical setting was demonstrated by analyzing plasma extracts from healthy volunteers as well as from pathological samples. While 5HTP was not present in any of the analyzed samples, the levels of 5HT and Trp in both normal and pathological plasma were determined.
Keywords: Serotonin; Tryptophan; 5-Hydroxytryptophan; Indolamines;

Using a typical HPLC assay, the characteristics of recovery, system precision and repeatability were investigated over a wide concentration range. In the presence of a constant amount of typical tablet excipients, the antidiabetic drug glibenclamide was analyzed in the range from 0.24 to 0.005 mg/mL (18 concentration levels, 6 independent sample preparations each). On the basis of a typical concentration for an HPLC glibenclamide assay of 0.2 mg/mL, this corresponds to a relative amount of 120–0.025% label claim. In the range from 120 to 0.075%, the recovery was found to be quite constant and systematically heightened mainly due to the evaporation from vials during centrifuging and the displacement of solvent volume by the added matrix. Both system precision and repeatability remain almost constant in the interval from 120 to 10% at a R.S.D.% of 0.31 and 0.70%, respectively, indicating that the sample preparation is the major error source in this range (0.63%). Between 10 and 0.25%, a linear relationship between the logarithmized concentration and the repeatability was noted. However, for lower amounts close to the limit of quantitation, the R.S.D.% of measurements increases much more distinctly. This increase is caused by a strong rise of the system precision. At this concentration range, system precision and repeatability are not significantly different any longer. This leads to the conclusion that with the injection error being constant the peak integration error becomes the dominating error source at low concentrations, e.g. at concentrations below the five-fold of the LOQ. The results obtained here agree well with earlier published data. As the quantitation limit of 0.05% can be regarded as typical for a pharmaceutical impurity control test, generalizations of these findings from this extensive data set should be possible. In this context, peak integration and improvements of the signal-to-noise ratio are the most promising measures to improve an unsatisfactory precision in LC.
Keywords: Wide concentration range; Analytical uncertainty; Recovery; Precision; Error structure;

Lipids (fatty acids) at a concentration range of 10–100 μg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10–230 μg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.
Keywords: Lipids; Cell culture medium;

Development and validation of a liquid chromatographic–tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine by Nicola Celli; Barbara Mariani; Luana K. Dragani; Stefania Murzilli; Cosmo Rossi; Domenico Rotilio (129-136).
Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography–electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5–1000 ng/ml; r ≥ 0.9968), intra- and inter-batch precision and accuracy (≤14.5%), and recoveries (94–106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.
Keywords: Caffeic acid phenethyl ester; Bioavailability;

In this article, an antibiotic, lincomycin was determined in the urine sample by microchip capillary electrophoresis (CE) with integrated indium tin oxide (ITO) working electrode based on electrochemiluminescence (ECL) detection. This microchip CE–ECL system can be used for the rapid analysis of lincomycin within 40 s. Under the optimized conditions, the linear range was obtained from 5 to 100 μM with correlation coefficient of 0.998. The limit of detection (LOD) of 3.1 μM was obtained for lincomycin in the standard solution. We also applied this method to analyzing lincomycin in the urine matrix. The limit of detection of 9.0 μM was obtained. This method can determine lincomycin in the urine sample without pretreatment, which demonstrated that it is a promising method of detection of lincomycin in clinical and pharmaceutical area.
Keywords: Microchip capillary electrophoresis; Lincomycin;

Determination of glimepiride in human plasma using semi-microbore high performance liquid chromatography with column-switching by Yun-Kyoung Song; Jeong-Eun Maeng; Hye-Ryung Hwang; Jeong-Sook Park; Bae-Chan Kim; Jin-Ki Kim; Chong-Kook Kim (143-149).
A fully automated semi-microbore high performance liquid chromatographic (HPLC) method with column-switching using UV detection was developed for the determination of glimepiride from human plasma samples. Plasma sample (900 μl) was deproteinated and extracted with ethanol and acetonitrile. The extract (70 μl) was directly injected into a Capcell Pak MF Ph-1 pre-column where the primary separation occurred to remove proteins and retain drugs using a mixture of acetonitrile and 10 mM phosphate buffer (pH 2.18) (20:80, v/v). The analytes were transferred from the pre-column to an intermediate column using a switching valve and then subsequently separated on an analytical column and monitored with UV detection at 228 nm. Glimepiride was eluted with retention time 34.9 min without interference of endogenous substance from plasma. The limit of quantification (LOQ) was 10 ng/ml for glimepiride. The calibration curves were linear over the concentration range of 10–400 ng/ml (r 2 = 0.9997). Moreover, inter- and intra-day precisions of the method were less than 15% and accuracies were higher than 99%. The developed method was successfully applied for the quantification of glimepiride in human plasma and was used to support a human pharmacokinetic study following a single oral administration of 2 mg glimepiride.
Keywords: Glimepiride; Column-switching; Bioavailability;

Analysis of non-covalent aggregation of synthetic hPTH (1–34) by size-exclusion chromatography and the importance of suppression of non-specific interactions for a precise quantitation by Marika Kamberi; Paul Chung; Richard DeVas; Lily Li; Zengji Li; Xiaoyan Ma (Sharon); Steven Fields; Christopher M. Riley (151-155).
There are few methods available for the rapid and precise quantitation of non-covalent aggregation. Size-exclusion chromatography (SEC), a traditional approach, used to measure the non-covalent aggregation can easily disrupt the weak forces holding an aggregate together. Under the conditions described in this paper the disaggregation of non-covalent aggregate of the synthetic human parathyroid hormone hPTH (1–34) due to hydrophobic/electrostatic interactions with the size-exclusion chromatography column packing was completely suppressed. This report details the effectiveness of adding salts and organic solvents in the mobile phase to overcome non-specific interactions that disrupt the aggregate during the SEC process and may aid in the understanding precise quantitation of non-covalent aggregation.
Keywords: SEC; PTH; Non-covalent aggregation;

We present a method for the analysis of urinary 16(5α)-androsten-3α-ol together with 5β-pregnane-3α,20α-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5αA), 5β-androstane-3α,17β-diol (5βA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6‰, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual δ13C-values for 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol are less than 0.9‰. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50 mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5α)-androsten-3α-ol and 5β-pregnane-3α,20α-diol, whereas the isotopic ratio values of 5β-pregnane-3α,20α-diol vary by more 5‰ upon ingestion of pregnenolone. We have observed δ13C-value changes lower than 1‰ for 16(5α)-androsten-3α-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5β-pregnane-3α,20α-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.
Keywords: Androstenol; Pregnenolone; Isotope ratio mass spectrometry (IRMS); Doping control;

The solubility product equilibrium constant for the precipitation between baicalin and berberine was investigated because of the current interest in decocting process of complex prescription of Chinese herbal medicine. HPLC was used for determining two compounds’ equilibrium concentrations at different precipitate conditions to calculate thermodynamic constants and study kinetic process. The analysis was performed on a Kromasil C18 column with TEA-adjusted 0.02 mol/L H3PO4 (pH 4.82)–acetonitrile (75:25) as mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. According to the experiment result, the molar ratio of baicalin and berberine in precipitate is about 1:1. The experimental K sp values are (1.01 ± 0.12) × 10−9  mol2/L2 at 20 °C in 0.02 mol/L NaH2PO4–Na2HPO4 (pH 4.82), and (3.20 ± 0.46) × 10−9  mol2/L2 at 40 °C in the same buffer. The precipitate reaction is an exothermic process and occurs immediately, even though the precipitate cannot be observed in time because the precipitate is light, yellow, flocculous and suspending in the yellow solution.
Keywords: Precipitate reaction; Baicalin; Berberine;

Simple and sensitive fluorimetric liquid chromatography method for the determination of valproic acid in plasma by Ming-Chun Lin; Hwang-Shang Kou; Cheng-Chung Chen; Shou-Mei Wu; Hsin-Lung Wu (169-172).
A simple and sensitive liquid chromatographic method is described for the analysis of valproic acid in human plasma. The method is based on the derivatization of valproic acid extracted from acidified plasma with 2-(2-naphthoxy)ethyl 2-(piperidino)ethanesulfonate. The resulting derivative is highly responsive to a fluorimetric detector (excitation at 230 nm and emission at 350 nm), giving a low detection limit of 0.6 μM (S/N = 3, 10 μl injected). The relative standard deviations of the method for intra- and inter-day analyses (n = 5) are below 3.3 and 4.1%, respectively. Toluene was used for the extraction of valproic acid from plasma and the toluene extract obtained was subjected to subsequent derivatization without solvent replacement. The simple method was applied to the analysis of valproic acid in plasma of dosed patients using only small amount of sample (10–50 μl plasma).
Keywords: Valproic acid;