Journal of Chromatography B (v.831, #1-2)

News Section (N1-N2).

A high performance liquid chromatographic assay for the quantitative determination of apomorphine in human plasma is described. Sample clean-up and concentration was optimised using solid-phase extraction on C18 cartridges, enabling rapid and sensitive determination of apomorphine and potential metabolites. The limit of apomorphine quantification, using fluorescence detection, was 0.5 ng/mL. The assay was stability-indicating, and allowed the detection of analytes in the presence of commonly co-administered anti-Parkinsonian drugs. Apomorphine was stable in frozen plasma containing 0.14% (w/v) ascorbic acid for 98 days, and through four freeze-thaw cycles. The assay has been used in clinical pharmacokinetic studies of apomorphine in patients with Parkinson's disease, and in preliminary studies of novel apomorphine delivery devices in volunteers.
Keywords: Apomorphine; Parkinson's disease; HPLC; Solid phase extraction; Plasma; Pharmacokinetics;

Quantification of biotin in feed, food, tablets, and premixes using HPLC–MS/MS by Ulrich Höller; Frédérique Wachter; Christof Wehrli; Christian Fizet (8-16).
Two sensitive and specific methods for quantification of biotin in feed, food, tablets, and premixes based on HPLC–MS/MS have been developed and validated. Depending on sample matrix and biotin content different extraction procedures and HPLC conditions were applied. Key steps in sample preparation were an alkaline extraction or a hydrolysis with sulphuric acid followed by enzymatic digest with papain. For many samples with low biotin content the latter combination of extraction steps was shown to be necessary for an optimal release of biotin from the matrix. The first time synthesis of deuterated biotin for use as internal standard allowed the compensation of losses during sample work-up and ion suppression during HPLC–MS/MS analysis. The new methods are faster than the commonly used microbiological assay using Lactobacillus plantarum. Additionally, they have a higher specificity as results for biotin are based on determination of a chemically defined compound, and not of a biological activity. Quantification is applicable to samples with a biotin content >100 μg/kg. Results obtained with the new methods have been compared with those of the microbiological assay, and were in good agreement.
Keywords: Quantitative biotin analysis; Feed; Food; Deuterated biotin; HPLC–MS/MS; Microbiological assay; Lactobacillus plantarum;

A novel and sensitive method for the determination of difenidol hydrochloride has been established using capillary electrophoresis coupled with end-column electrogenerated chemiluminescence (ECL) detection, based on the ECL reaction of tris(2,2′-bypyridine)ruthenium(II) (Ru(bpy)3 2+) with the tertiary amino groups of the difenidol analyte. Parameters that affect separation and detection were optimized. Calibration curve was linear over the range from 1 × 10−6  M to 6 × 10−5  M with a detection limit of 1 × 10−7  M (S/N = 3). Separation of difenidol hydrochloride from clomifene citrate and lidocaine was achieved using the proposed method. This method was successfully utilized to the assay of the active ingredients of the “difenidol hydrochloride” tablets and to the investigation on the interaction of difenidol hydrochloride with hemoglobin. The number of binding sites and the binding constant were estimated as (11.2 and 2.5) × 103  M−1, respectively.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Difenidol hydrochloride; Hemoglobin;

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K d of the ligand–sorbate complex is approximately 1 × 10−6 and 0.4 × 10−5  M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.
Keywords: Lectin affinity chromatography; Biospecific adsorbents; Concanavalin A; Glucose oxidase; Adsorption isotherms;

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites by Sabine Engelhardt; Ingolf Meineke; Jürgen Brockmöller (31-35).
Torasemide is a “loop type” diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C2-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10 mg oral dose during a 24 h study interval were located at 1 h for torasemide, 1 h for M1 and 2 h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.
Keywords: Torasemide; Loop type diuretics; HPLC; Solid-phase extraction; Pharmacokinetics;

Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry by Rui Wang; GuangJi Wang; Haiping Hao; HaiTang Xie; Jinfeng Zhang; Feng Wu (36-41).
Atractylenolide III is a major active component in Atractylodes macrocephala. This paper describes a simple, rapid, specific and sensitive method for the quantification of atractylenolide III in rat plasma using a liquid–liquid extraction procedure followed by liquid chromatography mass spectrometric (LC-MS) analysis. A Kromasil 3.5 μm C18 column (150 mm × 2.00 mm) was used as the analytical column. Linear detection responses were obtained for atractylenolide III concentration ranging from 5 to 500 ng mL−1. The precision and accuracy data, based on intra-day and inter-day variations over 5 days were within 10.29%. The lower limit of quantitation for atractylenolide III was 5 ng mL−1, using 0.1 mL plasma for extraction and its recoveries were greater than 85% at the low, medium and high concentrations. The method has been successfully applied to a pharmacokinetic study in rats after an oral administration of atractylenolide III with a dose of 20.0 mg kg−1. With the lower limits of quantification at 5 ng mL−1 for atractylenolide III, this method was proved to be sensitive enough for the pharmacokinetics study of atractylenolide III.
Keywords: Atractylenolide III; LC-MS; Pharmacokinetics;

Quantitative analysis of trimethylsilyl derivative of hydroxyurea in plasma by gas chromatography–mass spectrometry by Hutchinson James; Masoud Nahavandi; Melville Q. Wyche; Robert E. Taylor (42-47).
Hydroxyurea is an antitumor drug widely used in the treatment of sickle cell disease. The drug has been analyzed in biological fluids by a number of high-performance liquid chromatography (HPLC) methods. This paper describes a fast and highly reliable capillary gas chromatography–mass spectrometry (GC–MS) procedure that was developed for the detection and quantitation of hydroxyurea in plasma. The compound and its labeled internal standard were liquid extracted from plasma and derivatized with BSTFA before analysis. The detection limit of the assay was 0.078 μg/ml and the limit of quantitation was 0.313 μg/ml with linearity up to 500 μg/ml. Intra-day variation, as coefficient of variation (C.V., %) over the selected concentration range, was 0.3–8.7% and inter-day variation was 0.4–9.6%.
Keywords: Hydroxyurea; Sickle cell disease; Liquid extraction; Quantitation; Derivatization; Gas chromatography–mass spectrometry;

Thalidomide enantiomers: Determination in biological samples by HPLC and vancomycin-CSP by Susan F. Murphy-Poulton; Frances Boyle; Xiao Qing Gu; Laurence E. Mather (48-56).
Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (−)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2 M), and stored at −80 °C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1–20 μg/ml, assay precision was 1–5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 μg/ml with 0.2–0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9 l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.
Keywords: Thalidomide stereoisomers; Enantioselective pharmacology; Cancer models; Pharmacokinetics;

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD+) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.
Keywords: Single-cell analysis; Electrochemical detection; Lactate dehydrogenase; Isoenzyme;

Analysis of cardiolipin in human muscle biopsy by Vladimir B. Ritov; Elizabeth V. Menshikova; David E. Kelley (63-71).
Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H2O:H2SO4) containing 0.5–1.0 mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5 mM H3PO4 as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5–1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5–40 pmol. The assay is linear over the range between 0.3 and 3.0 mg of tissue (R 2  = 0.998). The assay provides good reproducibility and accuracy (within 5–10%).
Keywords: Cardiolipin; Mitochondria; Skeletal muscle; Human; Fluorescent derivatization; HPLC; 1-Pyrenyldiazomethane;

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring—Comparison of HPLC with UV-, single MS- and tandem MS-detection by Jochen Tuerk; Marius Reinders; Dennis Dreyer; Thekla K. Kiffmeyer; Klaus Gerhard Schmidt; Heinz-Martin Kuss (72-80).
Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD = 1–2 ng/cm2). Samples from biological monitoring of occupational uptake should be analyzed by LC–MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 μg/L for LC–MS and 0.01 to 0.9 μg/L for LC–MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm2.
Keywords: Liquid chromatography-mass spectrometry; Antibiotics; Sulfonamides; β-Lactames; Fluoroquinolones; Wipe Samples; Urine; Environmental and biological monitoring; Occupational exposure;

In order to investigate how the α1-acid glycoprotein (AGP) concentrations of neonates change in response to surgical stress, a simple high-performance liquid chromatography (HPLC)-assay for the measurement of α1-acid glycoprotein levels was developed. A fraction containing α1-acid glycoprotein was isolated from the bulk of plasma protein by addition of 0.6 M perchloric acid and was then analysed directly on a short PLRP-S 4000 Å reversed phase column column. The method was validated by analysis of pooled plasma from healthy adults both in comparison with a calibration curve and by standard additions. The procedure was able to isolate α1-acid glycoprotein rapidly (<30 min) and required only 50 μl of plasma. The mean extraction recovery was 79.1% (CV 6.4%). The within-run precision for the analysis of three replicates of quality control sample ranged from ±1.2 to ±3.8% and the between-run precision was ±6.1%. The method was linear (r 2  = 0.988) over a concentration range from 6 to 100.0 mg/100 ml. The AGP levels in neonatal samples ranged from 25 to 93 mg/100 ml.
Keywords: Anaesthetics; Alpha1-acid glycoprotein; HPLC; Neonates; Plasma;

A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC–electrospray mass spectrometry. Following liquid–liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.
Keywords: Omeprazole; Metabolites; LC–MS;

Liquid chromatography–electrospray mass spectrometry determination of free and total concentrations of ropivacaine in human plasma by Olivier Mathieu; Dominique Hillaire-Buys; Christophe Dadure; Franck Barnay; Jean Claude Mathieu-Daudé; Françoise Bressolle (91-98).
A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry (LC–ESI–MS) method was developed for the determination of free and total ropivacaine in human plasma. The work-up procedure involved a simple precipitation of plasma proteins with methanol. Etidocaine served as the internal standard. After microscale equilibrium-dialysis, measurement of free ropivacaine levels was performed after direct injection of the dialysate into the chromatograph. The system used a Zorbax eclipse XD8 C8 analytical column packed with 5 μm diameter particles as the stationary phase. The mobile phase consisted of a 15-min gradient (mobile phase A: 0.05% (v/v) trimethylamine in acetonitrile, mobile phase B: 2 mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 275 for ropivacaine and m/z 277 for etidocaine. The drug/internal standard peak area ratios (plasma) or peak areas (dialysate) were linked via a quadratic relationship to concentrations. Precision ranged from 1 to 7.6% and accuracy was between 92.6 and 109%. The lower limits of quantitation were 1 μg/l in plasma and 2 μg/l in the dialysate. This method was found suitable for the analysis of plasma samples collected during a clinical trial performed in 30 infants undergoing epidural anaesthesia or continuous psoas compartment block.
Keywords: Ropivacaine; Local anaesthetics; Equilibrium-dialysis; Plasma; Quantitation LC–ESI–MS;

A solid-phase extraction (SPE) method was developed using 8 M urea to desorb and extract organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs) from avian serum for analysis by capillary gas chromatography with electron capture detection (GC–ECD). The analytes were efficiently extracted from the denatured serum–lipoprotein–analyte complex by one passage through an Oasis® hydrophilic–lipophilic-balanced (HLB) SPE cartridge. No further clean-up was necessary, the entire extraction procedure and GC–ECD analysis can be accomplished in less than 3 h. Serum volumes ranged from 100 μL to 1 mL with absolute recoveries of 90–101% for PCBs and 74% to 101% for the OC pesticides.
Keywords: Serum; Polychlorinated biphenyls; Organochlorine pesticides; SPE; Urea; Oasis®;

Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood by Kiyotaka Usui; Takanori Hishinuma; Hiroaki Yamaguchi; Naoki Tachiiri; Junichi Goto (105-109).
Rapid and reliable methods for identification of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) in antiseptic and hemolyzed blood using electrospray ionization mass spectrometry (ESI-MS) were developed. Fragmental analysis provides accurate evidence for the presence of CHD in the samples. For the determination of CHD in hemolyzed blood, the method was also developed using LC-ESI-MS. Linearity of calibration curve was obtained over the concentration range of 0.1–11 μg/mL with residuals from −4.3 to 6.7%. We applied the methods to the case of suicidal injection of antiseptic and successfully detected CHD and NPEOn from hemolyzed blood. The CHD concentration was 352 μg/mL.
Keywords: Chlorhexidine; Nonylphenolethoxylates; LC-ESI-MS; Human; Blood; Forensic science; Forensic medicine; LC-ESI; Disinfectant; Toxicology; Suicide; Antiseptic;

Breast milk is one possible route of exposure to environmental chemicals, including phenols and chlorinated organic chemicals for breast-fed infants. We developed a highly sensitive method of analyzing breast milk for triclocarban (3,4,4′-trichlorocarbanilide) and eight phenolic compounds: bisphenol A (BPA), 4-tert-octylphenol (4-tOP), ortho-phenylphenol (OPP), 2,4-dichlorophenol, 2,5-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, and 2-hydroxy-4-metoxybenzophenone (BP-3). The method includes adding a solution containing a stable isotope of each chemical, enzymatic hydrolysis of the conjugated chemicals in the milk, and on-line solid-phase extraction coupled with high performance liquid chromatography–tandem mass spectrometry. It can also be used to measure the free (unconjugated) species by omitting the enzymatic deconjugation step. The method, validated using pooled breast milk samples, has inter-day coefficient of variations ranging from 4.8 to 18.9% for most analytes, and spiked recoveries generally about 100%. Detection limits for most analytes are below 1 ng/mL in 100 μL of breast milk. We tested the usefulness of the method by measuring concentrations of these nine compounds in 20 breast milk samples. BPA, OPP, and BP-3 were detected in more than 60% of the samples tested. The free species of these compounds appear to be most prevalent in milk.

Characterization of immunoaffinity purified peptidoglycan-associated lipoprotein of Actinobacillus actinomycetemcomitans by Riikka Ihalin; Maribasappa Karched; Kjell Eneslätt; Sirkka Asikainen (116-125).
Peptidoglycan-associated lipoprotein (PAL) is a highly conserved structural outer membrane protein among Gram-negative bacteria. In some species, it is proinflammatory and released extracellularly. We purified a newly identified PAL (AaPAL) of a periodontal pathogen Actinobacillus actinomycetemcomitans by using AaPAL antipeptide antibodies coupled to immunoaffinity chromatography column. No protein impurities originating in A. actinomycetemcomitans were found in the final product. Sera from patients infected by A. actinomycetemcomitans recognized the purified AaPAL. The present purification method seems to be suitable for isolation of AaPAL and probably PALs of other bacterial species, and applicable in studies investigating proinflammatory mechanisms of A. actinomycetemcomitans.
Keywords: Peptidoglycan-associated lipoprotein; Actinobacillus actinomycetemcomitans; Immunoaffinity chromatography; Antipeptide antibodies;

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry–positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 μL of a urine sample were diluted with 3 mL of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5–1.0 ng/μL, and we made the calibration curves to be linear from 0.25 to 250 ng/μL (r 2  > 0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.
Keywords: Polyol-pathway; Urine; Gas chromatography/mass spectrometry–positive chemical ionization; Sorbitol; Glucose;

The development of a validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionisation (ESI(+)) and multiple reaction monitoring (MRM) for the selective and sensitive bioanalytical determination of amisulpride, a substituted benzamide derivative, in human plasma is described. Plasma was cleaned up using a liquid–liquid extraction (diisopropylether:dichloromethane = 1:1 (v/v)) procedure. The chemically related drug sulpiride was used as internal standard (ISTD) and a primary calibration function was established in the concentration range of 0.50–500.52 ng/ml for amisulpride in plasma by triple analysis of the corresponding calibration standards. A linear relationship between concentration and signal intensity (given as peak area ratio analyte/ISTD) was obtained (linear regression: r  = 0.9999). A lower limit of quantification (LLQ) of 0.50 ng/ml was used during measurement of study plasma samples. Satisfying results of within-day precision (CV = 0.79 to 1.98%) and accuracy (mean relative deviation: −1.68 to 3.58%) and between-day precision (CV = 1.34 to 4.62%) and accuracy (mean relative deviation: −1.73 to −3.77%) as well as of recovery (amisulpride: 81.74 to 84.83%; sulpiride: 58.65%) and selectivity investigations confirmed the high reliability of this established LC-MS/MS method. Sufficient stability of amisulpride in plasma was achieved during freeze–thaw-cycles, for storage periods of 24 h at room temperature and 20 days at <−20 °C as well as in extracts (storage conditions: <−20 °C, 6 days and 7 °C, 6 days) with mean relative deviations between − 2.83 and 2.91%. An example of a pharmacokinetic profile determined after administration of an amisulpride 200 mg dose in a pilot study is given in this paper. A peak plasma concentration (C max) of 522.58 ng/ml was achieved after 3.55 h (t max). Corresponding values of areas under the plasma concentration–time curve (AUC) of 3405.35 ng h/ml (AUC0−∞) and 3306.54 ng h/ml (AUC0−tlast) were obtained. The terminal plasma elimination half-life (t 1/2) was 10.36 h.
Keywords: Amisulpride; Sulpiride; Benzamide derivative; Antipsychotic drug; Human plasma; Liquid chromatography; Tandem mass spectrometry; Positive electrospray ionisation;

Simultaneous determination of four active alkaloids from a traditional chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC–MS–MS by Hui-Liang Li; Wei-Dong Zhang; Run-Hui Liu; Chuan Zhang; Ting Han; Xiang-Wei Wang; Xiao-Lin Wang; Jian-Bao Zhu; Chun-Lin Chen (140-146).
A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 μm, 100 × 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 μm, 20 × 4 mm). The method was validated with the concentration range 1–1000 ng/mL in plasma and 10–1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC–MS–MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.
Keywords: Corydalis saxicola Bunting.; Dehydrocavidine; Coptisine; Dehydroapocavidine; Tetradehydroscoulerine; HPLC; LC–MS–MS; Pharmacokinetics;

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[14C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0–100 μg/mL for zebularine, 2.5–50 μg/mL for uridine, 1.0–10 μg/mL for uracil and 0.5–5.0 μg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.
Keywords: Zebularine; Metabolism; Murine; Radioactivity; DNA methyltransferase inhibitor;

SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-d-galactose (ΔDi-0S), 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose (ΔDi-4S), and 2-acetamido-2-deoxy-3-O-(β-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose (ΔDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-β-d-gluco-4-enepyranosyluronic acid)-d-galactose (ΔDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C18 column (4.6 mm × 150 mm, 5 μm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2 mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r 2  ≥ 0.994) in the concentration range 1.0-20 μg/mL with a lower limit of quantification (LLOQ) of 1.0 μg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of −8.92 to 5.61% and −16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.
Keywords: Glycosaminoglycans; Disaccharides; Biomarkers; Anti-thrombin; Pharmacodynamics;

Ion-pair reversed-phase HPLC: Assay validation of sodium tanshinone IIA sulfonate in mouse plasma by S.J. Mao; S.X. Hou; Z. Liang; Y.Q. Bi; Y. Wu; H. Li; H. Jin (163-168).
Sodium tanshinone IIA sulfonate (STS), a hydrophilic ionic substance, is used as a cardiovascular drug. An ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method for the determination of STS in mouse plasma was initially developed. The assay involved a rapid and simple extraction process and subsequent detection at 271 nm. The retention time for STS was 7.5 min. Based on extracted STS standard mouse plasma at 1.5,10 and 50 μg/ml, the assay precision were 2.7, 2.1 and 1.7% with a mean accuracy of 96.7, 98.5 and 99.4%, respectively. At plasma concentration of 1.5, 50 and 75 μg/ml, the mean recovery of STS were 93.1, 96.3 and 97.5%. The limit of detection (LOD) and limit of quantification (LOQ) for STS was 0.1 μg/ml and 0.5 μg/ml, respectively. Linear responses were observed over a wide concentration range (0.5–100 μg/ml) for STS in mouse plasma. STS can be detected after intravenous administration. This method was performed for the first time in pharmacokinetic studies of STS in the mouse.
Keywords: Sodium tanshinone IIA sulfonate (STS); Ion-pair; Reversed-phase HPLC; Pharmacokinetics;

Determination of lapatinib (GW572016) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC–ESI-MS/MS) by Feng Bai; Burgess B. Freeman; Charles H. Fraga; Maryam Fouladi; Clinton F. Stewart (169-175).
A sensitive method for the determination of lapatinib (GW572016) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Plasma samples (100 μL) were prepared using solid phase extraction (SPE) columns, and 6.0 μL of the reconstituted eluate was injected onto a Phenomenex® CuroSil-PFP 3 μ analytical column (50 mm × 2.0 mm) with an isocratic mobile phase. Analytes were detected with a PE SCIEX API-365 LC–MS/MS system at unit (Q1) and low (Q3) resolution in positive multiple reaction monitoring mode (m/z 581 (precursor ion) to m/z 364 (product ion) for lapatinib). The mean recovery for lapatinib was 75% with a lower limit of quantification of 15 ng/mL (S/N = 11.3, CV ≤ 14%). This method was validated over a linear range of 100–10,000 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma lapatinib concentrations in a Phase I study in children with cancer.
Keywords: Plasma; Laptinib (GW572016); Liquid chromatography; Electrospray tandem mass spectrometry (LC–ESI-MS/MS);

The validation of a pre-column derivatization procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to the determination of the amino acid content by RP-HPLC with fluorescence detection (λ excitation 250 nm, λ emission 395 nm) in milk-cereal based infant foods was carried out. The analytical parameters: linearity (0.0025–0.2 mM), precision of the method (0.2–3.5% variation coefficients), accuracy (derivatization: 86–106% average recovery and method: 88.3–118.2% average recovery) and the limits of detection (0.016–0.367 μM) and quantification (0.044–1.073 μM) were determined. Glutamic acid, proline and leucine were the most abundant amino acid whereas the lowest contents corresponded to tyrosine and cysteine.
Keywords: Infant foods; Amino acids; RP-HPLC; AQC;

All azo colorants whose metabolism can liberate a carcinogenic arylamine, are suspected of having carcinogenic potential. Therefore, a new azo compound 4-phenethyl-5-[4-(1-(2-hydroxyethyl)-3,5-dimethyl-4-pyrazolylazo)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (substrate) was prepared to investigate its in vitro and in vivo biotransformation in rats by HPLC. Chromatographic separation of substrate and its metabolites was performed using a Chromasil C18 column. The mobile phase consisted of acetonitrile and water in a linear gradient system. From the biotransformation of this compound, the reduction metabolite 4-(2-phenethyl)-5-(4-aminophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione was identified by comparing it to reference standard by HPLC-DAD. In the in vivo study, identification of the unknown peak which was the N-acetylation metabolite was confirmed by LC–MS spectrometry. Besides this, the azo compound was reduced to its corresponding amine in intestinal and cytosolic parts. In addition, oxidation of the methyl group and the phenyl ring, and reduction of azo group to hydrazo were identified in the cytosolic part using LC–MS.
Keywords: Azo; Metabolism; HPLC;

Capillary electrophoresis screening of poisonous anions extracted from biological samples by Robert Gillette; Janet M. Doyle; Mark L. Miller; Madeline A. Montgomery; George W. Mushrush (190-195).
A method was developed for screening human biological samples for poisonous anions using capillary electrophoresis (CE) employing indirect UV detection. The run buffer consisted of 2.25 mM pyromellitic acid, 1.6 mM triethanolamine, 0.75 mM hexamethonium hydroxide and 6.5 mM NaOH at pH 7.7. Biological samples were pretreated using solid phase extraction. The method was applied to the analysis of human blood, plasma, urine, and intestinal contents. Twenty-nine different anions were detectable at aqueous concentrations of 1 part per million (ppm) with a typical analysis time less than 20 min. Intraday migration time R.S.D. and peak area R.S.D. for blood samples were less than 1.1% and 6.3%, respectively. Interday migration time R.S.D. for plasma samples ranged from 7.5% to 10.4%. The new method produced efficient separations of various target anions extracted from complex biological matrices.
Keywords: Capillary electrophoresis; Anions; Screening; Poison; UV detection; Urine; Blood; Plasma; Intestinal contents;

Sample stacking for the analysis of penicillins by microemulsion electrokinetic capillary chromatography by Patricia Puig; Francesc Borrull; Carme Aguilar; Marta Calull (196-204).
We present a method for determining eight penicillin antibiotics using microemulsion electrokinetic chromatography (MEEKC). We studied how the composition of the microemulsion affected separation by modifying such parameters as the surfactant or the addition of organic solvents. The best microemulsion system consisted of 0.5% ethyl acetate, 1.2% 1-butanol, 2% Brij 35, 10% 2-butanol and 86.3% 10 mM borate buffer at pH 10. We studied the suitability of this microemulsion composition for analyzing a commercial drug. To improve the sensitivity of the method, we used the stacking technique reversed electrode polarity stacking mode (REPSM), which increased the detection limits by about 40-fold.
Keywords: Antibiotics; Capillary electrophoresis; Penicillins; Microemulsion electrokinetic chromatography; Preconcentration;

Use of microbore LC–MS/MS for the quantification of oxcarbazepine and its active metabolite in rat brain microdialysis samples by Katrien Lanckmans; Ralph Clinckers; Ann Van Eeckhaut; Sophie Sarre; Ilse Smolders; Yvette Michotte (205-212).
A microbore LC–MS/MS method is developed and validated for the quantification of the anti-epileptic drug oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxycarbamazepine (MHD) in rat brain microdialysates, together with the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide (m-CBZ). The benefits of gradient versus isocratic separation are shown, next to the improved sensitivity resulting from the addition of 0.1% formic acid to the mobile phase. The coupling of microdialysis with ESI-MS requires sample desalting for which column switching was applied. Using weighed regression to calculate the calibration curves (1–1000 ng/mL), the assay was validated in terms of linearity, accuracy and precision, yielding a sensitive (limit of quantification is 1 ng/mL) and selective method for quantification of OXC, MHD and m-CBZ. By applying this method, we were able to determine the extracellular concentrations of OXC and MHD during at least 4 h after intraperitoneal (i.p.) administration of 10 mg/kg OXC.
Keywords: Oxcarbazepine; 10,11-Dihydro-10-hydroxycarbamazepine; Microbore LC–MS/MS; Column switching; Rat brain microdialysis;

A 96-well single-pot protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of muraglitazar, a PPAR α/γ dual agonist, in human plasma. The internal standard, a chemical analogue, was dissolved in acetonitrile containing 0.1% formic acid. The solvent system was also served as a protein precipitation reagent. Human plasma samples (0.1 mL) and the internal standard solution (0.3 mL) were added to a 96-well plate. The plate was vortexed for 1 min and centrifuged for 5 min. Then the supernatant layers were directly injected into the LC/MS/MS system. The chromatographic separation was achieved isocratically on a Phenomenox C18(2) Luna column (2 mm × 50 mm, 5 μm). The mobile phase contained 20/80 (v/v) of water and acetonitrile containing 0.1% formic acid. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API 3000. The standard curve, which ranged from 1 to 1000 ng/mL, was fitted to a 1/x weighted quadratic regression model. This single-pot approach effectively eliminated three time consuming sample preparation steps: sample transfer, dry-down, and reconstitution before the injection, while it preserved all the benefits of the traditional protein precipitation. By properly adjusting the autosampler needle offset level, only the supernatant was injected, without disturbing the precipitated proteins in the bottom. As a result, the quality of chromatography and column life were not compromised. After more than 600 injections, there was only slightly increase of column backpressure. The validation results demonstrated that this method was rugged and provide satisfactory precision and accuracy. The method has been successfully applied to analyze human plasma samples in support of a first-in-man study. This method has also been validated in monkey and mouse plasma for the determination of muraglitazar.
Keywords: Single-pot protein precipitation; LC/MS/MS; Muraglitazar;

Dialkylphosphates (DAP) are urinary markers of the exposure to organophosphates pesticides. The aim of this study was to develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantitative determination of the following DAP: dimethylphosphate (DMP), dimethythiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Dibutylphosphate (DBP) was used as internal standard. This method was based on a liquid–liquid extraction procedure, a chromatographic separation using an Inertsil ODS3 C18 column and mass spectrometric detection in the negative ion, multiple reaction monitoring (MRM) mode, following two ion transitions per compound. It yielded a limit of quantification of 2 μg/L for the six compounds and intra-assay coefficients of variation (CV%) lower than 20%. This method was applied to the analysis of urines samples from a small cohort of non-exposed volunteers. At least one of the six DAP was detected in each sample. This result confirmed the feasibility of a LC–MS/MS procedure for monitoring the general population exposure to some frequently employed organophosphate pesticides.
Keywords: Dialkylphosphates; Organophosphorus pesticides; Urine; Liquid chromatography; Tandem mass spectrometry;

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2–200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.
Keywords: Propiverine; Column switching; High-performance liquid chromatography (HPLC);

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.
Keywords: Mitochondrial disease; Mitochondrial DNA; Mutation discovery; Heteroplasmy; Heteroduplex analysis; Denaturing HPLC; dHPLC;

To facilitate the assessment of drug safety and determination of phamacokinetics, an anion exchange isolation of zidovudine triphosphate (ZDV-TP) from human peripheral blood mononuclear cells (hPBMC), coupled with dephosphorylation, desaltation, and detection by liquid chromatography–tandem mass spectroscopy (LC–MS–MS) was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular ZDV-TP was produced. ZDV-TP was isolated from ZDV, ZDV-monophosphate (ZDV-MP), and ZDV-diphosphate (ZDV-DP), which were all present in the cell lysate, by performing a salt gradient anion exchange SPE. Isolated ZDV-TP was dephosphorylated with acid phosphatase to its parent drug form, ZDV. ZDV was then desalted and concentrated for tandem mass spectral detection. An LC–MS–MS methodology was developed and validated for the determination of molar ZDV directly corresponding to the intra-hPBMC molar ZDV-TP concentration. ZDV-TP concentrations were determined in femtomoles per million hPBMCs (fmol/106  cells). The assay was able to determine ZDV-TP concentrations accurately and precisely within the range of 5–640 fmol/106  cells with 10 million cells per sample analyzed. Inter- and intra-day accuracy and precision data for back calculated standards and quality controls fell within 15% of nominal. The assay correlated well with a previous ELISA method developed and validated in our laboratory, and has been successfully used to quantitate ZDV-TP concentrations in patients being routinely monitored and treated with ZDV.
Keywords: LC–MS–MS; Zidovudine-triphosphate; hPBMC;

Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography by Stefania Notari; Alessio Bocedi; Giuseppe Ippolito; Pasquale Narciso; Leopoldo Paolo Pucillo; Gianna Tossini; Raffaele Perrone Donnorso; Francesco Gasparrini; Paolo Ascenzi (258-266).
Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC–UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 μL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 μL methanol. Twenty microliters of these samples were injected into a HPLC–UV system, the analytes were eluted on an analytical C18 Symmetry™ column (250 mm × 4.6 mm I.D.) with a particle size of 5 μm. The mobile phase (0.01 M KH2PO4 and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 μg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005–10 μg/mL) in plasma has been studied at 24.0 °C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC–UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.
Keywords: HIV protease inhibitors; HIV nucleoside reverse transcriptase inhibitors; HIV non-nucleoside reverse transcriptase inhibitors; HPLC–UV; Therapeutic drug monitoring;

Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC–ESI–MS/MS by Mariella Zoppa; Lorena Gallo; Franco Zacchello; Giuseppe Giordano (267-273).
In our study we have developed an HPLC–ESI–MS/MS method for qualitative and quantitative analysis of underivatized amino acids on dry blood spots. The sensitive and specific instrumental performances permitted the chromatographic separation of 40 amino acids and their isomers within 10 min. The method has been set up for cases of suspected metabolic diseases revealed by newborn screening. What is new is that it is applied on the same blood spots used for newborn screening, instead of plasma, in order to avoid involvement of doctors, increased anxiety for parents, stress for patients for plasma collection, long time of waiting and further costs for analysis.
Keywords: Newborn screening; Blood spot; Underivatized amino acids; HPLC–ESI–MS/MS;

Determination of succinylacetone in dried blood spots and liquid urine as a dansylhydrazone by liquid chromatography tandem mass spectrometry by Osama Y. Al-Dirbashi; Mohamed S. Rashed; Herman J. Ten Brink; Cornelis Jakobs; Najlaa Filimban; Lujane Y. Al-Ahaidib; Minnie Jacob; Moeen M. Al-Sayed; Zuhair Al-Hassnan; Eissa Faqeih (274-280).
Succinylacetone (SA) is a specific marker for the inherited metabolic disease, hepatorenal tyrosinemia. We developed a stable-isotope dilution liquid chromatography tandem mass spectrometry for the determination of SA in dried blood spots (DBS) and liquid urine using a 13C4-SA as internal standard. SA was extracted, converted to the butyl ester and derivatized with dansylhydrazine (Dns-H). Calibration curves in DBS and urine calibrators were linear up to 100 and 30 μM, respectively. At a signal-to-noise ratio of 3, the limits of detection in DBS and urine were 0.2 and 0.005 μM, respectively. Total run time was 5 min. Intra- and inter-assay precision expressed as coefficient of variation were better than 9.1% with more than 96% recovery. The method was applied retrospectively and prospectively for the diagnosis of hepatorenal tyrosinemia and for follow-up of patients under treatment.
Keywords: Hepatorenal tyrosinemia; Tyrosine; Succinylacetone; Dansylhydrazine; Tandem mass spectrometry; Newborn screening;

Selected ion monitoring in quantitative gas–liquid chromatographic – mass spectrometric detection of fatty acid methyl esters from environmental samples by Merja Kontro; Leena Korhonen; Terttu Vartiainen; Päivi Pellikka; Pertti J. Martikainen (281-287).
To calculate selected ion monitoring (SIM) gas–liquid chromatography (GLC)–mass spectrometry (MS) results of phospholipid fatty acids (PLFAs) from environmental samples, coefficients were calculated for each fatty acid by dividing the sum of ion intensities in SCAN with that of ions followed in SIM. The SIM chromatogram areas were multiplied with the coefficients, and then processed as in SCAN. The results were compared to those obtained using calibration curves and SCAN. The calibration curve and coefficient based results had the greatest errors of 7.8 and 6.7%, respectively, outside standard deviations of SCAN percentages. The PLFA contents calculated using calibration curves and coefficients were 104.9 ± 7.3% and 101.5 ± 8.6%, respectively, of SCAN values. SIM increased sensitivity approximately 10-fold from SCAN, and the smallest detectable injected amount was approximately 50 ng (0.18 nmol) for 20 fatty acids, corresponding to 4 × 106 cells.
Keywords: GLC–MS; SIM; SCAN; Fatty acids; Environment;

On-line identification of the constituents of Buyang Huanwu decoction in pig serum using combined HPLC–DAD–MS techniques by Donghui Yang; Shaoqing Cai; Hongyu Liu; Xinxin Guo; Changling Li; Mingying Shang; Xuan Wang; Yuying Zhao (288-302).
Buyang Huanwu decoction (BYHWD) is a widely used Chinese traditional compound medicine that has proved effective in treating cerebrovascular illnesses; however, its active substances have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), photodiode-array detection and mass-spectrometry techniques were used to study the constituents of BYHWD from pig serum after oral administration. A total of 45 characteristic HPLC peaks were detected from serum containing drug. The chemical structures of nine of the peaks were tentatively elucidated as 7,3′-dihydroxy-4′-methoxyisoflavone-7-O-glucuronide (P1), 7-hydroxy-4′-methoxyisoflavone-7-O-glucuronide (P2), 7,2′,4′-trihydroxy-3′-methoxyisoflavane-7-O-sulphate (P3), 3-hydroxy-9,10-dimethoxypterocarpan-3-O-glucuronide (P4), 7,2′-dihydroxy-3′,4′-dimethoxyisoflavane-7-O-glucuronide (P5), 3-hydroxy-9,10-dimethoxypterocarpane-3-O-sulphate (P6), 4(1H)-quinolinone (P7 or P8), 4-hydroxyquinoline (P8 or P7) and oleic acid (P9). All of the identified peaks, with the exception of P9, were metabolites of the constituents of BYHWD in vivo.
Keywords: Buyang Huanwu decoction; Serum containing drug; HPLC–DAD–MS; Metabolite;

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of p-coumaric acid in rat plasma and applied to a pharmacokinetic study in rats after administration of a prodrug, E-6-O-p-coumaroyl scandoside methyl ester, isolated from Hedyotis diffusa (Willd.). Sample preparation involved protein precipitation with acetonitrile. The supernatant was then injected onto a DiamonsilTM C18 column (250 mm × 4.6 mm i.d., 5 μm). The mobile phase consisted of acetonitrile–water (21:79, v/v) with 1% glacial acetic acid. The UV detector was set at 310 nm. The lower limit of quantification of p-coumaric acid in rat plasma was 0.02 μg/mL. The calibration curves were linear over the concentration range 0.02–5 μg/mL with correlations greater than 0.999. The assay procedure was applied to the study of the metabolite pharmacokinetics of E-6-O-p-coumaroyl scandoside methyl ester in rat.
Keywords: E-6-O-p-Coumaroyl scandoside methyl ester; p-Coumaric acid; Pharmacokinetics; Metabolite;

Based on the theory of stochastic resonance, the signal to noise ratio (SNR) of HPLC/UV chromatographic signal of roxithromycin is enhanced by cooperation of signal, noise and nonlinear system. A simple new method for the determination of low concentration of roxithromycin in beagle dog plasma is presented. Using signal enhancement by stochastic resonance, this method extends the limit of quantitation from the reported 0.5 to 0.1 μg/ml. During validation of the new method, HPLC/MS was used as a comparison technique. The results indicate that the recovery and low concentrations of roxithromycin in beagle dog plasma were equivalent between the two methods (P  > 0.05). Stochastic resonance may be a promising tool for improving detection limits in trace analysis.
Keywords: Stochastic resonance; Roxithromycin; Weak signal; HPLC/UV; HPLC/MS;

The GC–MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC–MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH]+ ions with little or no fragmentation. In the case of carboxylic acids, [M + 18]+ ions predominated but [MH]+ ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC–MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH]+ ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.
Keywords: tBDMS; Amino acids; Polyamines; Urea; Sulfur amino acids; Dipeptides; GC–MS; Ammonia PCI; Trimethyl tin; Amino acid release;

For a microdialytic trapping method we systematically investigated changes in concentrations of 2,5-dihydroxy-benzoic acid (2,5-DHBA) and 2,3-dihydroxy-benzoic acid (2,3-DHBA) in freshly prepared solutions of salicylic acid (SA). The solvent was 0.9% saline exposed to different atmospheric concentrations of oxygen (0, 21, and 100%). The solutions were treated by freezing–thawing and an ultrasonic bath in presence and absence of aluminium foil. Without aluminium the concentrations of 2,5-DHBA and 2,3-DHBA kept constant over an observed period of 160 min on different levels from below 20 ng/ml to about 100 ng/ml. In presence of aluminium the concentrations increased to maximum 307 ng/ml after 160 min. Ultrasonic irradiation amplified this effect to maximum 341 ng/ml. HPLC/ECD processing and quantitative analysis of dihydroxy-benzoic acids (DHBAs) in microdialysis may be artificially influenced by varying oxygen environment and metal catalysis.
Keywords: Salicylate trapping method; 2,5-DHBA; 2,3-DHBA; Hyperoxia; Oxygen; Metal catalysis;

Longitudinal profiling of urinary steroids by gas chromatography/combustion/isotope ratio mass spectrometry: Diet change may result in carbon isotopic variations by Christophe Saudan; Matthias Kamber; Giulia Barbati; Neil Robinson; Aurélien Desmarchelier; Patrice Mangin; Martial Saugy (324-327).
Longitudinal profiling of urinary steroids was investigated by using a gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method. The carbon isotope ratio of three urinary testosterone (T) metabolites: androsterone, etiocholanolone, 5β-androstane-3α,17β-diol (5β-androstanediol) together with 16(5α)-androsten-3α-ol (androstenol) and 5β-pregnane-3α,20α-diol (5β-pregnanediol) were measured in urine samples collected from three top-level athletes over 2 years. Throughout the study, the subjects were living in Switzerland and were residing every year for a month or two in an African country. 13C-enrichment larger than 2.5‰ was observed for one subject after a 2-month stay in Africa. Our findings reveal that 13C-enrichment caused by a diet change might be reduced if the stay in Africa was shorter or if the urine sample was not collected within the days after return to Switzerland. The steroids of interest in each sample did not show significant isotopic fractionation that could lead to false positive results in anti-doping testing. In contrast to the results obtained with the carbon isotopic ratio, profiling of urinary testosterone/epitestosterone (T/E) ratios was found to be unaffected by a diet change.
Keywords: Steroids; Diet; Isotope ratio mass spectrometry (IRMS); Doping control;

A sensitive and specific liquid chromatography–electrospray ionization-mass spectrometry (LC–ESI-MS) is described for quantitation of salbutamol in human urine using nadolol as the internal standard (I.S.). Urine samples were hydrolyzed with β-glucuronidase followed by a solid-phase extraction procedure using Bond Elut-Certify cartridges. The HPLC column was an Agilent Zorbax SB-C18 column. A mixture of 0.01 M ammonium formate buffer (pH 3.5)–acetonitrile (85:15, v/v) was used as the mobile phase. Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. Selected ion monitoring (SIM) mode was used to monitor m/z 166 for salbutamol and m/z 310 for I.S. Good linearity was obtained in the range of 10.0–2000.0 ng/ml. The limit of quantification was 10.0 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 7.3%. The accuracy as determined from QC samples was within ±2.6%. The method was applied for determining excretion curves of salbutamol.
Keywords: Salbutamol; Urine; Antidoping control; Liquid chromatography–mass spectrometry;

Author Index (335-337).

Keyword Index (338-345).