Journal of Chromatography B (v.878, #24)

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair by Elissandro Soares Emídio; Vanessa de Menezes Prata; Fernando José Malagueño de Santana; Haroldo Silveira Dórea (2175-2183).
A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 25−1 fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg−1 (CBD and CBN) and 20–500 pg mg−1 (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg−1 and 1–20 pg mg−1, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg−1 (CBD), 20–232 pg mg−1 (THC) and 9–107 pg mg−1 (CBN), confirming the suitability of the method for monitoring studies.
Keywords: Cannabinoids; Hollow fiber liquid-phase microextraction; Fractional factorial design; GC–MS; Hair;

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C18 column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL−1 for baicalin and 0.08832 to 4.416 μg mL−1 for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL−1 for baicalin and 0.04416 μg mL−1 for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the C max1 was 1.64 ± 0.99 and 5.66 ± 2.02, the t max1 was 0.13 ± 0.05 and 0.20 ± 0.07, the C max2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the t max2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t 1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the C max1 was 1.39 ± 0.83 and 1.45 ± 0.37, the t max1 was 0.21 ± 0.20 and 0.17 ± 0.01, the C max2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the t max2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference (p  < 0.05) was observed for t 1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.
Keywords: Baicalin; Wogonoside; Pharmacokinetics; Liquid chromatography; Mass spectrometry;

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads by Aya Mizutani; Kenichi Nagase; Akihiko Kikuchi; Hideko Kanazawa; Yoshikatsu Akiyama; Jun Kobayashi; Masahiko Annaka; Teruo Okano (2191-2198).
For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.
Keywords: Thermo-responsive polymer; Poly(N-isopropylacrylamide); Atom transfer radical polymerization; Polymer brush; Poly(styrene-co-divinylbenzene); Thermo-responsive chromatography; Peptide;

Microscale analysis of amino acids using gas chromatography–mass spectrometry after methyl chloroformate derivatization by Wen-Ping Chen; Xiao-Yuan Yang; Adrian D. Hegeman; William M. Gray; Jerry D. Cohen (2199-2208).
To conduct studies of stable isotope incorporation and dilution in growing plants, a rapid microscale method for determination of amino acid profiles from minute amounts of plant samples was developed. The method involves solid-phase ion exchange followed by derivatization and analysis by gas chromatography–mass spectrometry (GC–MS). The procedure allowed the eluent to be derivatized directly with methyl chloroformate without sample lyophilization or other evaporation procedures. Sample extraction and derivatization required only ca. 30 min and quantification of the 19 amino acids eluted from the cation exchange solid-phase extraction step from a single cotyledon (0.4 mg fresh weight) or three etiolated 7-day-old Arabidopsis seedlings (0.1 mg fresh weight) was easily accomplished in the selected ion monitoring mode. This method was especially useful for monitoring mass isotopic distribution of amino acids as illustrated by Arabidopsis seedlings that had been labeled with deuterium oxide and 15N salts. Sample preparation was facile, rapid, economical, and the method is easily modified for integration into robotic systems for analysis with large numbers of samples.
Keywords: Arabidopsis; GC–MS; SPE column; Stable isotope; Metabolite profiling; Alkyl chloroformate;

Simultaneous HPLC–MS–MS quantification of 8-iso-PGF and 8,12-iso-iPF in CSF and brain tissue samples with on-line cleanup by Magdalena Korecka; Christopher M. Clark; Virginia M.-Y. Lee; John Q. Trojanowski; Leslie M. Shaw (2209-2216).
Quantitation of isoprostanes such as 8-iso-PGF and 8,12-iso-iPF-VI in biological fluids has been proposed as a reliable test of oxidant stress and inflammation in a variety of disorders. This paper presents a liquid chromatography method with tandem mass spectrometry detection for the simultaneous analysis of these two isoprostanes in human CSF and brain tissue samples. An API 5000 triple quadrupole instrument (AB Sciex, Foster City, CA, USA) with an APCI ion source was used in this study. Aliquots of CSF samples (0.25 mL) were treated with a methanol:zinc sulfate mixture followed by on-line cleanup on an extraction column (Validated-C18) with 0.1% formic acid. The brain tissue samples were homogenized and lipids were extracted using Folch solution. Solid-phase extraction columns (C18) were used for the purification of the brain isoprostane fraction. Chromatographic separation was achieved using an analytical column (Synergi C18 HydroRP) with 0.1% formic acid in water and a mixture of methanol:acetonitrile under isocratic conditions. The mass spectrometer was operated in the MRM scan and negative ion mode. The quadrupoles were set to detect the molecular ions [M−H] and high mass fragments of isoprostanes: m/z 353 → 193 amu (8-iso-PGF) and m/z 353 → 115 amu (8,12-iso-iPF-VI) and their deuterated internal standards: m/z 357 → 197 amu (8-iso-PGF-d4) and m/z 364 → 115 amu (8,12-iso-iPF-VI-d11). The lower limit of quantification was 2.5 pg/mL for 8-iso-PGF and 5.0 pg/mL for 8,12-iso-PF-VI for the CSF method and 10.0 pg/0.1 g of tissue and 30.0 pg/0.1 g of tissue for 8-iso-PGF and 8,12-iso-iPF-VI, respectively, for the brain tissue method. No ion suppression or enhancement of the detection of 8-isoPGF, 8,12-isoPF-VI or both internal standards was found.
Keywords: 8-iso-PGF; 8,12-iso-iPF-VI; HPLC–MS/MS; On-line cleanup; CSF; Brain tissue samples;

Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry by Jignesh M. Parekh; Rajendrasinh N. Vaghela; Dipen K. Sutariya; Mallika Sanyal; Manish Yadav; Pranav S. Shrivastav (2217-2225).
A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC–ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid–liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200 μL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50 mm × 4.6 mm, 3 μm) analytical column using isocratic mobile phase, consisting of 20 mM ammonium acetate–methanol (25:75, v/v), at a flow-rate of 0.8 mL/min. The precursor → product ion transition for teriflunomide (m/z 269.0 → 82.0) and IS (m/z 434.1 → 350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1–4001 ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0 min and is applied to a bioequivalence study of 20 mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.
Keywords: Teriflunomide; LC–ESI-MS/MS; Human plasma; Bioequivalence study;

Fibers with polypyrrole and polythiophene phases for isolation and determination of adrenolytic drugs from human plasma by SPME-HPLC by Pawel Olszowy; Malgorzata Szultka; Tomasz Ligor; Jacek Nowaczyk; Boguslaw Buszewski (2226-2234).
In this study, polypyrrole (PPy) and polythiophene (PTh) SPME coatings and their ability to extract selected adrenolytic drugs with different physico-chemical properties from standard solutions and human plasma samples were evaluated. In measurements metoprolol, oxprenolol, mexiletine, propranolol, and propaphenon were investigated. The main parameters such as extraction time, desorption conditions and pH influence were examined. Inter-day precisions were in range 0.1–2.0%, 1.1–2.9%, 1.3–2.6%, 0.1–2.6% and 0.3–2.1% for metoprolol, oxprenolol, mexiletine, propranolol and propaphenon, respectively. Accuracies were less than 15%, which was evaluated by analyzing preparation samples of five replicates. The method was successfully applied to human plasma samples spiked with selected adrenolytic drugs. The method was linear in the concentration range from 1 to 10 μg/ml for all of studied adrenolytic drugs using human plasma samples. The PTh-SPME coating displayed higher extraction efficiency towards the target analytes in comparison to PPy-SPME. The reproducibility of the extraction using polypyrrole and polythiophene fibers was confirmed by variation coefficients lower than 8% and 3%, respectively.
Keywords: Solid-phase microextraction; Electropolymerization; Adrenolytic drugs; Liquid chromatography;

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of goserelin in rabbit plasma by Min Kyung Kim; Tae Ho Lee; Joon Hyuk Suh; Han Young Eom; Jung Won Min; Hyesun Yeom; Unyong Kim; Hyung Joon Jung; Kyung Hoi Cha; Yong Seok Choi; Jeong-Rok Youm; Sang Beom Han (2235-2242).
A rapid and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry method (LC–ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis® HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 μL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0 mm × 150 mm, 5 μm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile = 85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250 μL/min. The LC–MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor–product ion transitions consisted of m/z 635.7 →  m/z 607.5 for goserelin and m/z 424.0 →  m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1–20 ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1 ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20 ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6 mg of goserelin in healthy white rabbits.
Keywords: Goserelin; Peptide; Liquid chromatography–tandem mass spectrometry; Rabbit plasma; Pharmacokinetic study;

Task-specific ionic liquid-assisted extraction and separation of astaxanthin from shrimp waste by Wentao Bi; Minglei Tian; Jun Zhou; Kyung Ho Row (2243-2248).
Astaxanthin, as an outstanding antioxidant reagent, was successfully extracted from shrimp waste by the ionic liquids based ultrasonic-assisted extraction. Seven kinds of imidazolium ionic liquids with different cations and anions were investigated in this work and one task-specific ionic liquid in ethanol with 0.50 mol L−1 was selected as the solvent. At the optimized ultrasonic extraction conditions, the extraction amount of astaxanthin increased 98% (92.7 μg g−1) compared to the conventional method (46.7 μg g−1). Furthermore, the extracted solution was isolated through the solid-phase extraction with a molecularly imprinted polymer sorbent. After loading the samples on molecularly imprinted polymer cartridge, the different washing and elution solvents, such as water, methanol, n-hexane, acetone and dichloromethane, were evaluated, and finally, astaxanthin was separated from the shrimp waste extract.
Keywords: Astaxanthin; Ionic liquid; Extraction; Separation;

Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing by Sascha Hagemann; Alexander Faude; Monika Rabenstein; Monika Balzer-Geldsetzer; Carmen Nölker; Michael Bacher; Richard Dodel (2249-2254).
Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way.
Keywords: Chromatofocusing; Affinity purification; Polyclonal immunoglobulin G; Two-dimensional gel electrophoresis;

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment by Olof Beck; Sören Sandqvist; Paul Eriksen; Johan Franck; Göran Palmskog (2255-2259).
At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.
Keywords: Abused drug testing; Liquid chromatography–mass spectrometry; Methadone; Human; Exhaled breath; Breath testing;

Determination of mitoxantrone in rat plasma by liquid chromatography–tandem mass spectrometry method: Application to a pharmacokinetic study by Peng Zhang; Guixia Ling; Jin Sun; Yongbing Sun; Xiaohui Pu; Zhiyuan Wang; Zhonggui He (2260-2265).
A rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed for quantification of mitoxantrone in rat plasma. The analyte and palmatine (internal standard) were extracted from plasma samples with diethyl ether–dichloromethane (3:2, v/v) and separated on a C18 column. The chromatographic separation was achieved within 2.5 min using methanol–10 mM ammonium acetate containing 0.1% acetic acid as the mobile phase at a flow rate of 0.2 mL/min. The method was linear over the range of 0.5–500 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. Finally, the method was successfully applied to a pharmacokinetic study of mitoxantrone in rats following intravenous administration.
Keywords: HPLC–MS/MS; Mitoxantrone; Plasma concentration;

Here we report a method capable of quantifying ginsenoside Rg3 in human plasma and urine. The method was validated over linear range of 2.5–1000.0 ng mL−1 for plasma and 2.0–20.0 ng mL−1 for urine using ginsenoside Rg1 as I.S. Compounds were extracted with ethyl acetate and analyzed by HPLC/MS/MS (API-4000 system equipped with ESI interface and a C18 column). The inter- and intra-day precision and accuracy of QC samples were ≤8.5% relative error and were ≤14.4% relative standard deviation for plasma; were ≤5.6% and ≤13.3% for urine. The Rg3 was stable after 24 h at room temperature, 3 freeze/thaw cycles and 131 days at −30 °C. This method has been applied to pharmacokinetic study of ginsenoside Rg3 in human.
Keywords: Ginsenoside Rg3; HPLC/MS/MS; Quantitative analysis;

A LC–MS/MS method was developed and validated for the determination of dehydroandrographolide succinate (DAS), a traditional Chinese medicine derivative used for the treatment of pneumonia, upper respiratory tract infection, and chronic bronchitis. Following protein precipitation, DAS was detected by ion transition at m/z 531.2/99.0 in multiple reaction monitoring mode with negative electrospray ionization-tandem mass spectrometry. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 10 ng/mL in human plasma. Good linearity was maintained over the range of 10–5000 ng/mL, and the correlation coefficient was better than 0.99. The accuracy ranged from 95.3% to 113%, RSD from 0.928% to 6.47%, for the within- and between-run analysis at all QC levels. The recovery ranged from 85.5% to 93.4% and the matrix effect from 107% to 119%. No significant carryover and good stability were found during method validation. The developed method was successfully applied to the determination of DAS in human plasma in a pharmacokinetic study following intravenous infusion of potassium sodium DAS to nine healthy volunteers.
Keywords: Dehydroandrographolide succinate; Protein precipitation; LC–MS/MS; Pharmacokinetic study; Human plasma;

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study by Prasad B. Phapale; Hae Won Lee; Mi-sun Lim; Sook Jin Seong; Eun-Hee Kim; Jeonghyeon Park; Miran Lee; Sung-Kyu Hwang; Young-Ran Yoon (2280-2285).
An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C18 column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.
Keywords: UPLC–MS/MS; Levosulpiride; Human plasma; Pharmacokinetic study; Bioanalytical method validation;

Simultaneous determination of isoniazid, rifampicin, levofloxacin in mouse tissues and plasma by high performance liquid chromatography–tandem mass spectrometry by Ping-Fei Fang; Hua-Lin Cai; Huan-De Li; Rong-Hua Zhu; Qin-You Tan; Wei Gao; Ping Xu; Yi-Ping Liu; Wen-Yuan Zhang; Yong-Chang Chen; Feng Zhang (2286-2291).
A rapid and selective high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for simultaneous determination of isoniazid (INH), rifampicin (RFP) and levofloxacin (LVX) in mouse tissues and plasma has been developed and validated, using gatifloxacin as the internal standard (I.S.). The compounds and I.S. were extracted from tissue homogenate and plasma by a protein precipitation procedure with methanol. The HPLC separation of the analytes was performed on a Welch materials C4 column (250 mm × 4.6 mm, 5.0 μm, USA) at 25 °C, using a gradient elution program with the initial mobile phase constituting of 0.05% formic acid and methanol (93:7, v/v) at a flow-rate of 1.0 ml/min. For all the three analytes, the recoveries varied between 83.3% and 98.8% in tissues and between 75.5% and 90.8% in plasma, the accuracies ranged from 91.7% to 112.0% in tissues and from 94.6% to 108.8% in plasma, and the intra- and inter-day precisions were less than 13.3% in tissues and less than 8.2% in plsama. Calibration ranges for INH were 0.11–5.42 μg/g in tissues and 0.18–9.04 μg/ml in plasma, for RFP were 0.12–1200 μg/g in tissues and 4.0–200 μg/ml in plasma, and for LVX were 0.13–26.2 μg/g in tissues and 0.09–4.53 μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11, 0.12 and 0.13 μg/g and for those in mouse plasma were 18.1, 20.0 and 21.8 ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04, 0.05 and 0.05 μg/g and for those in mouse plasma were 5.5, 6.0 and 6.6 ng/ml, respectively. The established method was successfully applied to simultaneous determination of isoniazid, rifampicin and levofloxacin in mouse plasma and different mouse tissues.
Keywords: Mass spectrometry; Tissue; Plasma; Isoniazid; Rifampicin; Levofloxacin;

A rapid, simple and sensitive HPLC–ESI–MS/MS method was developed for the simultaneous determination of capsaicin and dihydrocapsaicin in rat plasma. Plasma samples containing capsaicin, dihydrocapsaicin and phenacetin (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with mobile phase of acetonitrile/water (55:45, v/v) containing 0.1% formic acid (v/v) at a flow rate of 0.2 mL/min with an operating temperature of 25 °C. Quantification was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source by selected reaction monitoring (SRM) of the transitions at m/z 306–137 for capsaicin, m/z 308–137 for dihydrocapsaicin and m/z 180–110 for the IS. Linear detection responses were obtained for capsaicin and dihydrocapsaicin ranging from 1 to 500 ng/mL and the lower limits of quantitation (LLOQs) for the two compounds were 1 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 9.79% for the two analytes, while the deviations of assay accuracies were within ±10.63%. The average recoveries of the analytes were greater than 89.88%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of capsaicin and dihydrocapsaicin in rats after subcutaneous administration of capsaicin (natural, containing 65% capsaicin and 35% dihydrocapsaicin).
Keywords: Capsaicin; Dihydrocapsaicin; HPLC–MS/MS; Pharmacokinetics;