Journal of Chromatography B (v.810, #2)
News Section (N1-N2).
Normal phase liquid chromatography–electrospray ionization tandem mass spectrometry analysis of phospholipid molecular species in blood mononuclear cells: application to cystic fibrosis by Marco Malavolta; Fabio Bocci; Emanuele Boselli; Natale G. Frega (173-186).
The use of HPLC coupled on-line with a mass spectrometer is a very powerful tool in order to analyze intact PLs molecular species (PMS) without the need of derivatization, thus decreasing the risk of artifacts formation. A normal-phase HPLC–ESI-MS–MS method has been developed in order to study the human blood mononuclear cell PMS composition. This method was applied to characterize PMS from seven CF subjects and from seven age-matched healthy subjects. More than 140 phospholipid molecular species from phosphatidylethanolamine (PE), plasmalogen phosphatidylethanolamine (pPE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC) and sphingomyelin (Sph) were identified and compared. Differences between the two groups were found in pPE (p16:0/22:6), pPE (p18:0/22:6), PE (16:0/20:4) and PC (16:0/18:2) which were significantly lower in CF subjects and in PC (16:0/16:1) which was significantly higher in CF subjects.
Keywords: Phospholipids; HPLC; Mass spectrometry; Cystic fibrosis; Blood mononuclear cells;
Determination of catechins and catechin gallates in tissues by liquid chromatography with coulometric array detection and selective solid phase extraction by Kai On Chu; Chi Chiu Wang; Ching Yan Chu; Michael Scott Rogers; Kwong Wai Choy; Chi Pui Pang (187-195).
Catechins levels in organ tissues, particularly liver, determined by published methods are unexpectedly low, probably due to the release of oxidative enzymes, metal ions and reactive metabolites from tissue cells during homogenization and to the pro-oxidant effects of ascorbic acid during sample processing in the presence of metal ions. We describe a new method for simultaneous analysis of eight catechins in tissue: (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG) (Fig. 1). The new extraction procedure utilized a methanol/ethylacetate/dithionite (2:1:3) mixture during homogenization for simultaneous enzyme precipitation and antioxidant protection. Selective solid phase extraction was used to remove most interfering bio-matrices. Reversed phase HPLC with CoulArray detection was used to determine the eight catechins simultaneously within 25 min. Good linearity (>0.9922) was obtained in the range 20–4000 ng/g. The coefficients of variance (CV) were less than 5%. Absolute recovery ranged from 62 to 96%, accuracy 92.5 ± 4.5 to 104.9 ± 6%. The detection limit was 5 ng/g. This method is capable for determining catechins in rat tissues of liver, brain, spleen, and kidney. The method is robust, reproducible, with high recovery, and has been validated for both in vitro and in vivo sample analysis.
Simultaneous determination of free mycophenolic acid and its glucuronide in serum of patients under mycophenolate mophetil therapy by ion-pair reversed-phase liquid chromatography with diode array UV detection by Antonella Aresta; Francesco Palmisano; Carlo G. Zambonin; Paolo Schena; Giuseppe Grandaliano (197-202).
An high performance liquid chromatography (HPLC)–UV method for the simultaneous determination of the free forms of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) in human serum samples was developed for the first time.Chromatographic separation was performed on octadecylsilane based stationary phase in combination with a mobile phase of methanol/buffered tetrabutylammonium (TBA) salt mixture.Sample pretreatment consisted of an ultrafiltration step followed by clean-up/enrichment on a C18 solid-phase extraction (SPE) cartridge. Average recoveries of (99.7 ± 0.2)% and (64.1 ± 6.9)% for free MPA and MPAG, respectively, were estimated in the concentration range from 0.5 to 10 μg/ml. The within-day and between-days coefficients of variation were 0.4 and 0.8% for free MPA (0.1 μg/ml spiking level) and 0.8 and 1.6% for free MPAG (5 μg/ml spiking level), respectively. The linear ranges for free MPA and MPAG were 0.06–1 and 0.2–10 μg/ml, respectively. Detection limits of 4 and 17 ng/ml for free MPA and MPAG were estimated in spiked serum.The same HPLC method was also capable of a simultaneous determination of the total concentration of MPA and MPAG when coupled to a proper sample pretreatment step. The potential of the method is demonstrated by excretion kinetics measurement in serum of patients receiving MMF therapy.
Keywords: Mycophenolic acid; Mycophenolic acid glucuronide; Mycophenolate mophetil;
Quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA) and other retinoids in rat plasma by liquid chromatography with diode-array detection by Chunyu Wu; Vincent Njar; Angela Brodie; Michael Borenstein; Ivo Nnane (203-208).
A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10–5.0 μg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life (t 1/2) was 19.0 ± 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.
Keywords: All-trans-retinoic acid; Retinoic acid metabolism blocking agent; Reversed-phase HPLC;
Application of liquid chromatography/mass spectrometry and nuclear magnetic resonance to the identification of degradates of a novel insulin sensitizer in aqueous solutions by Michael H. Wang; Yunhui Wu; Tsang-Lin Hwang; Xue-Zhi Qin; Shyam Karki; Varaporn Treemaneekarn (209-219).
Degradation of a novel insulin sensitizer in aqueous solutions was studied using high pressure liquid chromatography/mass spectrometry (LC/MS). The insulin sensitizer, containing a thiazolidine-2,4-dione (TZD), was a new class of antidiabetic agent for the treatment of type II diabetes. Chemical stability of the insulin sensitizer was evaluated by stressing its aqueous solutions at 40 °C for 24 h. Oxygen was removed from one of the solutions by bubbling pure nitrogen through to identify non-oxidative pathways. LC/MS analyses of the stressed solutions revealed that hydrolysis and oxidation are the primary degradation pathways for the studied compound. A α-thiol acetic acid, acyl amide, and two dimeric diastereomers were the main degradates of the insulin sensitizer. The α-thiol acetic acid served as an intermediate-like species, and oxidized to two dimeric degradates upon exposing to air. All of them were identified as ring-opening products of the TZD. The entities of the acyl amide and dimeric degradates were respectively verified by a synthetic standard or NMR following isolation of a diastereomeric degradate. Characterization using MS in both positive and negative ion scans were discussed for an isolated diastereomeric degradate. Mechanisms of fragmentation and formation for those degradates are presented based on the MS result.
Keywords: Insulin sensitizer; Thiazolidine-2,4-dione; Degradation; Dimerization;
Determination of deltamethrin and its metabolite 3-phenoxybenzoic acid in male rat plasma by high-performance liquid chromatography by Yan Ding; Catherine A. White; S. Muralidhara; James V. Bruckner; Michael G. Bartlett (221-227).
The pyrethroid insecticide—deltamethrin [(S)-α-cyano-3-phenoxybenzyl-(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate] is widely used throughout the world in agricultural applications. It is also used to treat humans, thereby creating a possible public health concern. The toxic effects of deltamethrin in mammals include choreoathetosis, hyperexcitability and salivation. The principal mechanisms of metabolism for deltamethrin are ester hydrolysis and oxidation at the 4′ position of the terminal aromatic ring. Few studies have been conducted on the toxicokinetics of deltamethrin or its metabolites, mainly due to the lack of quick, sensitive, and validated analytical methods. In this study, we describe the first method to simultaneously determine deltamethrin and its major metabolite 3-phenoxybenzoic acid (3-PBAcid). This method utilizes protein precipitation and high-performance liquid chromatography to support a toxicokinetic study in the rat. The limit of quantitation for both deltamethrin and 3-PBAcid was 0.1 μg/ml. The recoveries for deltamethrin and 3-PBAcid were approximately 91 and 94%, respectively. Intra-day (n = 5) precision (relative standard deviation, %R.S.D.) and accuracy (%Error) for deltamethrin ranged from 1.5 to 12.3% and from 4.0 to 10.6%, respectively. The inter-day (n = 15) precision (%R.S.D.) and accuracy (%Error) for deltamethrin ranged from 7.1 to 11.6% and from 6.3 to 8.2%, respectively. Intra-day (n = 5) precision (%R.S.D.) and accuracy (%Error) for 3-PBAcid ranged from 1.7 to 13.1% and from 1.3 to 14.5%, respectively. The inter-day (n = 15) precision (%R.S.D.) and accuracy (%Error) for 3-PBAcid ranged from 5.4 to 14.5% and from 5.1 to 12.8%, respectively. Finally, this method was applied to a toxicokinetic study of deltamethrin in the rat.
Keywords: Pyrethroid pesticides; Deltamethrin; 3-Phenoxybenzoic acid;
Development and validation of a liquid chromatography–tandem mass spectrometric method for the determination of α-methyltyrosine in human plasma by David Humphries; Kenneth Ruterbories; Clark Chan; Rangaraj Narayanan (229-234).
A sensitive and selective LC–MS–MS method for the isolation and quantification of α-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC–MS–MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C18 column (2.0 mm × 50 mm; 5 μm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water–acetic acid (100:0.1, v/v) and acetonitrile–acetic acid (100:0.1, v/v). The structural analog α-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.
Keywords: α-Methyltyrosine; Validation;
Liquid chromatography–positive ion electrospray mass spectrometry method for the quantification of citalopram in human plasma by Constantinos Pistos; Irene Panderi; Julia Atta-Politou (235-244).
A rapid, sensitive and novel narrow-bore liquid chromatography–mass spectrometric method was developed and fully validated for the quantification of citalopram in human plasma. The analyte and internal standard (imipramine) were extracted by liquid–liquid extraction with a mixture of hexane–heptane–isopropanol (88:10:2, v/v/v). The use of a Hypersil BDS C8 micro-bore column (250 mm × 2.1 mm i.d.; 3.5 μm particle size), results in substantial reduction in solvent consumption. The mobile phase consisted of 10 mM ammonium formate–formic acid (pH 4.5) and acetonitrile (30:70, v/v), pumped at a flow rate of 0.15 ml min−1. The analytes were detected after positive electrospray ionization using the selected ion-monitoring mode of the species at m/z 325 for citalopram and m/z 281 for imipramine. The method had a chromatographic run time of 10.0 min and a linear calibration curve over the range 0.50–250 ng ml−1 (r 2 > 0.996). The limit of quantitation was 0.50 ng ml−1. Accuracy and precision were below the acceptance limits of 15%.
Keywords: Citalopram; Imipramine; High-performance liquid chromatography; Electrospray ionization; Mass spectrometry;
Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography by Hua Zhao; Kenji Hamase; Akiko Morikawa; Zongyin Qiu; Kiyoshi Zaitsu (245-250).
A sensitive and selective method for the determination of four threonine (Thr) isomers (l-Thr, d-Thr, l-allo-Thr and d-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of d-Thr, d-allo-Thr and l-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol–5 pmol per injection, and that of l-Thr is 50 fmol–50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of d-Thr, d-allo-Thr and l-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of d-Thr and d-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of d-Thr (0.85 ± 0.05 nmol/g wet tissue) and d-allo-Thr (5.01 ± 0.32 nmol/g wet tissue) were found in the corpus striatum. l-allo-Thr was not present in any of the tested tissues and physiological fluids.
Keywords: Chiral separation; Amino acid; HPLC; Threonine; allo-Threonine;
Development and validation of a method for the purity determination of (3β,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin by Babak Jamali; Steen G. Andersen; Helle Brødholt; Lene Wendel; Vibeke Bødstrup (251-257).
A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3β,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN® (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin.After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm.The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3–0.8% (depending on the impurity) corresponding to 1.9–5.1 ng. The limit of quantification was found to be 0.8–2.5% (depending on the impurity) corresponding to 5.2–16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100–129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50–150% of the nominal concentration (640 ng, injection volume is 100 μl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r 2) was 0.999 for FF-MAS (50–150% level) and 0.977–0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities.The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.
Keywords: FF-MAS; 3β,20R-4,4-Dimethylcholesta-8,14,24-trien-3-ol; Recombinant human albumin; RECOMBUMIN; Sample preparation; Validation;
Development and validation of a liquid chromatography–mass spectrometry method for the quantitation of naltrexone and 6β-naltrexol in guinea pig plasma by Satyanarayana Valiveti; Buchi N. Nalluri; Dana C. Hammell; Kalpana S. Paudel; Audra L. Stinchcomb (259-267).
A quantitative liquid chromatographic–electrospray ionization mass spectrometry method for the determination of naltrexone and 6β-naltrexol in guinea pig plasma has been developed and validated using naloxone as an internal standard. A single step precipitation-extraction technique was carried out to extract the plasma samples using acetonitrile:ethyl acetate (1:1, v/v). The chromatographic separation was performed on a C18 column using a mobile phase consisting of 35:65 (v/v) acetonitrile:2 mM ammonium acetate with 0.01 mM ammonium citrate at a flow rate of 0.25 mL/min. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The mean recoveries for naltrexone, naltrexol, and naloxone were 91.7, 89.3, and 99.0%, respectively. The lower limit of quantification (LLOQ) for naltrexone and 6β-naltrexol was 1.25 ng/mL, and the limit of detection (LOD) was 0.75 ng/mL. The method was applied to a pharmacokinetic study in order to assess the drug disposition of naltrexone in guinea pigs.
Keywords: Naltrexone; 6β-Naltrexol; Pharmacokinetics;
Determination of acetone in human breath by gas chromatography–mass spectrometry and solid-phase microextraction with on-fiber derivatization by Chunhui Deng; Jie Zhang; Xiaofeng Yu; Wei Zhang; Xiangmin Zhang (269-275).
Analysis of breath acetone has been used as a diagnostic tool for diabetes. Due to its nature of volatility and activity, it is very difficult to accurately measure the concentration of acetone in human breath by gas chromatography–mass spectrometry (GC–MS). To overcome this problem, we developed a new method using GC–MS and solid-phase microextraction (SPME) with on-fiber derivatization to determine acetone in human breath. Breath gas from controls and diabetic patients was collected in 3-l Tedlar bags. O-2,3,4,5,6-(Pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) in solution was firstly adsorbed on the SPME fiber of 65 μm polydimethylsiloxane–divinylbenzene (PDMS–DVB), and then the fiber was further headspace exposed in exhaled gas in the Tedlar bag at 40 °C for 4 min. Finally, the formed acetone oxime on the fiber was desorbed and analyzed by GC–MS. Using external standard method, acetone in the human breath was quantitatively analyzed by measurement of its oxime. The method provided a low detection limit of 0.049 ppbv for acetone in breath, relative standard deviation (R.S.D.) value of 3.4%, excellent accuracy. In addition, the method required simple sample preparation and no organic solvent. Acetone in diabetic breath was found to be higher than 1.71 ppmv, while its concentration in normal breath was lower than 0.76 ppmv. The results show that GC–MS and SPME with on-fiber derivatization is a simple, rapid and sensitive and solvent-free method for determination of low concentration acetone in breath and analysis of breath acetone can be used as supplementary tool for diagnosis of diabetes.
Keywords: Acetone; Breath analysis; Derivatization, GC;
Assay of plasma semicarbazide-sensitive amine oxidase and determination of its endogenous substrate methylamine by liquid chromatography by Hui Li; Wenhong Luo; Jijin Lin; Zhexuan Lin; Yuan Zhang (277-282).
Semicarbazide-sensitive amine oxidase (SSAO) is present in plasma, as well as in other tissues. Previous studies indicated that SSAO is of important physiological and pathophysiological functions. HPLC methods were developed for the assay of SSAO in plasma, and for the determination of plasma methylamine, an SSAO's endogenous substrate. Benzylamine was used as artificial substrate for the enzyme activity assay of SSAO. A 0.2-ml aliquot of plasma was incubated with benzylamine at 37 °C for 30 min. Benzaldehyde, the enzymatic reaction product, was derivatized with 2,4-dinitrophenylhydrazine (DNPH), and analyzed with HPLC and UV detection. SSAO enzyme activity is defined as benzaldehyde (nmol) formed per ml plasma per hour. Recoveries of benzaldehyde spiked to plasma were between 63.5 and 68.2% with relative standard deviation less than 3%. To determine methylamine in plasma, a 0.1-ml aliquot of plasma was deproteinized by trichloroacetic acid and centrifugation. The supernatant was derivatized with dansyl chloride and analyzed by HPLC with fluorescence detection. Recoveries of spiked methylamine at ppb (ng/ml) level were between 93.7 and 97.6% with relative standard deviation less than 2.5%.
Keywords: Semicarbazide-sensitive amine oxide; Methylamine; Enzymes;
Determination of Δ9-tetrahydrocannabinol from rabbit plasma by gas chromatography–mass spectrometry using two ionization techniques by Janne Mannila; Marko Lehtonen; Tomi Järvinen; Pekka Jarho (283-290).
The purpose of the study was to develop a gas chromatography–mass spectrometric (GC–MS) method for the identification and quantitation of Δ9-tetrahydrocannabinol (THC) in rabbit plasma. Two ionization techniques were utilized for GC–MS: electron impact ionization (EI) after i.v. administration and negative chemical ionization (NCI) after sublingual administration. THC was isolated from plasma by solid phase extraction and derivatized by either trimethylsilylation (EI) or trifuoroacetylation (NCI), with deuterated THC as an internal standard. The validity of analytical method was confirmed by investigating selectivity, limit of quantitation, linearity, accuracy, precision, recovery and stability of the analyte. The method proved to be selective, linear, accurate and precise over a range of 10–430 and 0.3–530 ng/ml of THC in plasma for EI and NCI, respectively. The extraction recovery was >81% for each concentration level studied, and the analyte was shown to be stable during storage and sample preparation. The method was applied successfully in analysing THC from rabbit plasma.
A new method for the quantitation of propofol in human plasma: efficient solid-phase extraction and liquid chromatography/APCI-triple quadrupole mass spectrometry detection by Lakshmikant Bajpai; Manoj Varshney; Christoph N. Seubert; Donn M. Dennis (291-296).
Propofol (2,6-diisopropyl phenol) is widely used for the induction and maintenance of anesthesia. Analyses of its pharmacokinetics require simple and sensitive methods for quantitation of propofol in human plasma. Previously reported HPLC and GC methods are limited by cumbersome extraction steps. We describe a novel method that combines sample preparation by solid-phase extraction (SPE) with hydrophilic–lipophilic balance cartridges and analysis with a sensitive LC-APCI-triple quadrupole mass spectrometry (MS/MS) method for better quantitation.The absolute recovery of the analyte was greater than 96%. The limit of quantification for propofol in plasma at a signal-to-noise ratio of 10 was 5 ng/ml. The precision of the assay yielded coefficients of variation ranging from 2.9 to 5.3% and an accuracies of 99–105%.Our method advances the quantitative analysis of propofol in human plasma by combining simple, rapid and efficient SPE with specific and sensitive quantitation by HPLC with APCI-MS/MS detection.
Keywords: Propofol; Human plasma; Solid-phase extraction; Liquid chromatography; Tandem mass spectrometry;
Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection by T.A.C. Vermeij; P.M. Edelbroek (297-303).
A rapid, simple and robust method is presented for the simultaneous determination of the γ-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP) and vigabatrin (VGB) in human serum by high-performance liquid chromatography (HPLC). Serum is deproteinized with trichloroacetic acid and aliquots of the supernatant are precolumn derivatized with o-phtaldialdehyde (OPA) and 3-mercaptopropionic acid. Separation is achieved on a Alltima 3C18 column using isocratic elution; the drugs are monitored using fluorescence detection. Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2% for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8 mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB. No interferences were found from commonly coadministered antiepileptic drugs (AEDs) and from endogenous amino acids.Experimental design in combination with statistical evaluation (ANOVA) was used to study the robustness of chromatography and sample preparation. The method is very suitable for routine therapeutic drug monitoring and for pharmacokinetic studies.
Keywords: Anticonvulsants; Pregabalin; Gabapentin; Vigabatrin; Drug analysis; HPLC; Validation; Design of experiments;
Determination of porphyrins and biliverdin in bile and excreta of birds by a single liquid chromatography–ultraviolet detection analysis by Rafael Mateo; Gloria Castells; Andy J. Green; Carlo Godoy; Carles Cristòfol (305-311).
Methods developed for porphyrin analysis have low recoveries and/or poor precision for the less polar protoporphyrin IX. We describe a simple method of analysis of porphyrins and biliverdin in bile and excreta of birds based on extraction with HCl 3N: acetonitrile and HPLC/UV analyses. Recoveries were good for protoporphyrin IX and other porphyrins (>79%). Applications of this method showed that porphyrins and biliverdin in birds excreta are mainly of biliary-fecal origin rather than urinary origin. Biliverdin and protoporphyrin IX increased proportionately more than the rest of the porphyrins and coproporphyrin III increased more than coproporphyrin I in the bile of Pb-poisoned mallards.
Keywords: Porphyrins; Biliverdin; Lead poisoning;
Bioanalysis of tobramycin for therapeutic drug monitoring by solid-phase extraction and capillary zone electrophoresis by Humphrey Fonge; Eliangiringa Kaale; Cindy Govaerts; Koenraad Desmet; Ann Van Schepdael; Jos Hoogmartens (313-318).
A method based on solid-phase extraction (SPE) and capillary zone electrophoresis (CZE) for the analysis of tobramycin in human serum is presented. An off-line SPE employing a carboxypropyl bonded phase (CBA) cartridge was used for the extraction of tobramycin from human serum. Adsorbed tobramycin was eluted from the CBA cartridge using a mixture of NH3 (25%, w/v)–methanol (30:70, v/v). After evaporation, the analyte was reconstituted and derivatized with o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA). The resulting tobramycin–OPA/MPA derivative was purified, and then identified by mass spectrometry. The tobramycin–OPA/MPA derivative was then analysed by CZE with a background electrolyte (BGE) comprising of 30 mM sodium tetraborate pH 10.0-acetonitrile (ACN) (80:20, v/v) with ultraviolet detection at 230 nm. A linear response was observed in the range of 0.3–30 μg/ml with r 2 = 0.992. The sensitivity of the method was determined by its limit of quantitation (LOQ) and limit of detection (LOD) of 0.3 μg/ml and 0.1 μg/ml, respectively. SPE recovery ranged from 68 to 79% at the trough levels to 98% at the peak levels found in serum. Furosemide has been added as internal standard (IS) to improve precision. For the therapeutic range of tobramycin in serum (2–10 μg/ml) the relative standard deviation (R.S.D.) was less than 11% for the entire SPE/CE process. The method demonstrated excellent selectivity as shown by the lack of interference from a total of 20 drugs investigated. The method was then used in therapeutic drug monitoring of patients receiving the drug.
Keywords: Tobramycin; Bioanalysis; Solid-phase extraction;
Development and validation of a method for the quantitation of Δ9tetrahydrocannabinol in oral fluid by liquid chromatography electrospray–mass-spectrometry by M. Concheiro; A. de Castro; O. Quintela; A. Cruz; M. López-Rivadulla (319-324).
Analysis of Δ9tetrahydrocannabinol (Δ9THC) and its metabolites in biological samples is of great relevance for forensic purposes. In the case of oral fluid (OF), the analysis should determine Δ9THC, whereas in urine, it detects the inactive metabolite tetrahydrocannabinol carboxylic acid (THC-COOH). Most laboratories analyze Δ9THC in such samples using GC–MS methods, but these procedures are time-consuming and involve unavoidable previous extraction and derivatization. No data is yet available on the application of liquid chromatography–mass-spectrometry to detect Δ9THC in oral fluid. We report a validation method in which the Δ9THC is isolated from oral fluid by a simple liquid–liquid extraction with hexane and subsequently analyzed by liquid chromatography–mass-spectrometry.The method here reported for the determination of Δ9THC in oral fluid only requires 200 μl of sample and achieves limits of detection of 2 ng/ml, and has been used to analyze oral fluid samples collected from current drug users.
Keywords: Δ9Tetrahydrocannabinol; Oral fluid; LC–MS;
One-step liquid chromatographic method for the determination of oxytetracycline in fish muscle by Rosie Coyne; Øivind Bergh; Ole Bent Samuelsen (325-328).
A one-step simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of oxytetracycline (OTC) in fish tissue. The method involves liquid extraction of muscle tissue, precipitation of proteins and reversed phase HPLC analysis with spectrophotometric detection. The limit of quantitation of OTC in spiked fish muscle was 0.04 μg/g and the method showed high linearity (r 2 = >0.999) in the working range of 0.04–2 μg/g. The precision (%R.S.D.) was between 1.9 and 7.5% for the concentration range 0.04–1.0 μg/g and there was no significant difference between the concentrations determined on three different test days for all four spiked concentrations. The percentage recovery over the spiked concentration range 0.04–1.0 μg/g was consistently within a narrow range of 33–35%. While the method had the advantage of high precision, sensitivity and linearity, the method's additional salient advantages included high sample through-put (60 individual preparations per day) and minimum amount of consumables, time and labour required to perform the analysis. The method was successfully applied to a pharmacokinetic study.
Keywords: Oxytetracycline; Fish;
Author Index to Vol. 810 (329-331).
Keyword Index to Vol. 810 (333-338).