Journal of Chromatography B (v.941, #C)

Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities, while its metabolic pathway in vivo has not been investigated. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the in vivo metabolism of irisolidone in rats. Plasma, bile, urine, and feces were collected from rats after a single 100 mg/kg oral dose of irisolidone. Protein precipitation, solid phase extraction (SPE) and ultrasonic extraction were used to prepare samples of plasma, bile/urine, and feces, respectively. A total of 46 metabolites were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The metabolic pathways of irisolidone in rats included decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation. The relative content of each metabolite was also determined to help understand the major metabolic pathways of irisolidone in rats.
Keywords: Irisolidone; Metabolic profile; UHPLC/Q-TOF MS; Metabolite; Rat;

Determination of allopurinol and oxypurinol in human plasma and urine by liquid chromatography-tandem mass spectrometry by Xia Liu; Xiao-Jia Ni; De-Wei Shang; Ming Zhang; Jin-Qing Hu; Chang Qiu; Fu-Tian Luo; Yu-Guan Wen (10-16).
Allopurinol is used widely for the treatment of gout, but its pharmacokinetics is complex and some patients show hypersensitivity, necessitating careful monitoring and improved detection methods. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of allopurinol and its active metabolite oxypurinol in human plasma and urine using 2,6-dichloropurine as the internal standard (IS). Analytes and the IS were extracted from 0.5 ml aliquots of plasma or urine using ethyl acetate and separated on an Agilent Eclipse Plus C18 column using methanol and ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid (95:5, v/v) as the mobile phase (A) for allopurinol or methanol plus 5 mM ammonium formate aqueous solution (95:5, v/v) as the mobile phase (B) for oxypurinol. Allopurinol was detected in positive ion mode and the analysis time was about 7 min. The calibration curve was linear from 0.05 to 5 μg/mL allopurinol in plasma and 0.5–30 μg/mL in urine. The lower limit of quantification (LLOQ) was 0.05 μg/mL in plasma and 0.5 μg/mL in urine. The intra- and inter-day precision and relative errors of quality control (QC) samples were ≤11.1% for plasma and ≤ 8.7% for urine. Oxypurinol was detected in negative mode with an analysis time of about 4 min. The calibration curve was linear from 0.05 to 5 μg/mL in plasma (LLOQ, 0.05 μg/mL) and from 1 to 50 μg/mL in urine (LLOQ, 1 μg/mL). The intra- and inter-day precision and relative errors were ≤7.0% for plasma and ≤9.6% for urine. This method was then successfully applied to investigate the pharmacokinetics of allopurinol and oxypurinol in humans.
Keywords: Allopurinol; Metabolite; HPLC-MS/MS; Human plasma; Pharmacokinetics;

Mass spectrometric investigation of chelerythrine and dihydrochelerythrine biotransformation patterns in human hepatocytes by Jan Vacek; Barbora Papoušková; Pavel Kosina; Adéla Galandáková; Jitka Ulrichová (17-24).
The quaternary benzo[c]phenanthridine alkaloids (QBAs) are an important subgroup of plant secondary metabolites. Their main representatives, sanguinarine (SG) and chelerythrine (CHE), have pleiotropic biological effects and a wide spectrum of medicinal applications. The biotransformation of SG and CHE has only been partially studied while subsequent oxidative transformation of their dihydro derivates, the main metabolites, is practically unknown. The aim of this study was to characterize the biotransformation of CHE and dihydrochelerythrine (DHCHE) in detail, with respect to their more extensive biotransformation than SG. Phase I as well as phase II biotransformation of both compounds was examined in human hepatocyte suspensions. Liquid chromatography with electrospray-quadrupole time-of-flight mass spectrometry (LC-ESI-QqTOF MS) was used for analysis of the metabolites. Using the LC-ESI-QqTOF MS method, we analyzed and then suggested the putative structures of 11 phase I and 5 phase II metabolites of CHE, and 11 phase I and 6 phase II metabolites of DHCHE. For the most abundant metabolites of CHE, DHCHE and O-demethylated DHCHE, their cytotoxicity on primary cultures of human hepatocytes was analyzed. Both metabolites were nontoxic up to 50 μM concentration and this indicates decreasing toxic effects for CHE biotransformation products, i.e. DHCHE and O-demethylated DHCHE.
Keywords: Chelerythrine; Dihydrochelerythrine; Benzo[c]phenanthridines; Metabolic transformation; Human hepatocytes;

Development and validation of a quantitative liquid chromatography tandem mass spectrometry assay for pristimerin in rat plasma by Xin Luan; Ying-Yun Guan; Ya-Rong Liu; Chao Wang; Mei Zhao; Qin Lu; Ya-Bin Tang; Xiao-Lin Wang; Chao Fang; Hong-Zhuan Chen (25-30).
A sensitive, rapid and simple LC–MS/MS analysis method was developed and validated for the determination of pristimerin (PR) in rat plasma. Protein precipitation with four volumes of acetonitrile as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Merck ZIC-HILIC column with the mobile phase of acetonitrile–water (containing 5 mM ammonium formate, pH 2.8) (85:15, v/v). PR (m/z 465.3–201.1) and glycyrrhetinic acid (internal standard, m/z 471.5–177.1) were monitored under positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of PR and IS was 2.45 min and 2.4 min, respectively. The limit of detection was 0.5 ng/mL and the linear range was 1–500 ng/mL. The intra-day and inter-day precision were 2.89–6.27% and 4.91–8.98%, and the intra-day and inter-day accuracy ranged from −5.81% to 8.64% and −7.37% to 9.57%, respectively. The matrix effects and absolute recovery ranged from 89.3% to 92.4% and 88.7% to 92.8%, respectively. The method has been successfully applied to the determination of PR concentration in rat plasma after intravenous administration (0.5 mg/kg).
Keywords: Pristimerin; HPLC–MS/MS; Pharmacokinetics; Rat plasma;

In vitro metabolism of alisol A and its metabolites’ identification using high-performance liquid chromatography–mass spectrometry by Yue Yu; Zhenzhen Liu; Ping Ju; Yuanyuan Zhang; Lunhui Zhang; Kaishun Bi; Xiaohui Chen (31-37).
A liquid chromatography–mass spectrometry (LC–MS) method was developed and successfully applied to the study on the enzyme kinetics of alisol A in rat liver microsomes (RLM) and human liver microsomes (HLM) incubation systems, and employed for semi-quantitative determination of each metabolite of alisol A. The metabolites of alisol A in RLM, HLM and human recombinant CYP3A4 enzyme incubation systems were identified by high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–QTOF MS). A total of 3 and 6 oxidative metabolites were found in RLM and HLM incubation systems, respectively. 3 metabolites found in both incubation systems were identified. The exact position of hydroxylation for the metabolites M1 and M2 could not be determined. Chemical inhibitors of cytochrome P450 (CYP450) and individual human recombinant CYP450 enzyme were used to identify the CYP450 isozymes involved in the formation of each metabolite of alisol A. The result indicated that the formation of each metabolite of alisol A was mainly catalyzed by CYP3A4 enzyme.
Keywords: Alisol A; Metabolism; HPLC–QTOF MS; Rat liver microsomes; Human liver microsomes;

A new kind of quercetin molecularly imprinted polymer (MIP) was synthesized and applied as a selective sorbent in matrix solid-phase dispersion (MSPD) for the extraction of quercetin in Herba Lysimachiae. The MIP was prepared by surface imprinting method using quercetin as template, methacrylic acid as functional monomer, trimethylolpropane trimethacrylate as crosslinker and methanol as porogen. The selectivity of quercetin MIP was evaluated according to their recognition to quercetin and a compound with similar molecular size (bergenin). Good binding for quercetin was observed in MIP adsorption experiment. The isothermal adsorption and dynamic adsorption experiments were also carried out in this study. The best quercetin extraction conditions were as follows: the ratio of MIP to sample was 1:1, the dispersion time was 10 min, washing solvent was 2% aqueous methanol and elution solvent was acetic acid–methanol (2:98, v/v). The proposed method was compared with the method used in Chinese pharmacopeia. The similar extraction yield was obtained by the two methods. Moreover, this method is faster, simpler and can realize extraction and purification procedures in the same system.
Keywords: Matrix solid-phase dispersion; Molecularly imprinted polymer; Quercetin; Herba Lysimachiae;

Click aspartic acid as H ILIC SPE material for selective enrichment of N-linked glycopeptides by Xiuling Li; Guobin Shen; FeiFang Zhang; Bingcheng Yang; Xinmiao Liang (45-49).
A novel hydrophilic interaction chromatography (HILIC) material was developed via clicking aspartic acid onto silica gel (termed as Click AA). The material demonstrated highly hydrophilic property and was used as solid phase extraction sorbent for selective enrichment of glycopeptides. By optimization of extraction conditions, Click AA exhibited high selectivity to glycopeptides with various peptide length and different types of glycans, which was much superior to similar commercial products. The application of Click AA to simulated proteomic samples further proved its specialty toward glycopeptides.
Keywords: Glycopeptides; Hydrophilic interaction chromatography; Aspartic acid; Click chemistry; Solid phase extraction;

Changes in volatile compounds of human urine as it ages: Their interaction with water by Jae Kwak; Claude C. Grigsby; Brittany R. Smith; Mateen M. Rizki; George Preti (50-53).
The urinary odors are commonly perceived as unpleasant. While numerous studies have identified the volatile organic compounds (VOCs) released from urine, the odorants responsible for the urine odor are not well characterized. Furthermore, anecdotal reports suggest that the odor of aged urine is different from that of fresh urine. However, no study has yet to investigate the specific VOCs released from aged urine. In this study, we analyzed and compared the VOCs released from fresh and aged urine samples, investigating the changes in the urinary VOCs as urine aged. We found an overall decrease in concentration of many urinary VOCs, and concluded this was due to the urine evaporating as it aged. On the contrary, some highly water-soluble compounds such as short and branched-chain organic acids and trimethylamine, increased. Their increased release is most likely due to the loss of water and the subsequent release of water-soluble VOCs as urine ages. We suggest that these VOCs may contribute to the odor of the aged urine.
Keywords: Urine; Volatile organic compounds (VOCs); Hydration status; Gas chromatography–Mass spectrometry (GC–MS); Metabolite Differentiation and Discovery Lab (MeDDL);

Two mixtures of decarboxylated and dehydrogenated betacyanins from processed red beet roots (Beta vulgaris L.) juice were fractionated by high performance counter-current chromatography (HPCCC) producing a range of isolated components. Mixture 1 contained mainly betacyanins, 14,15-dehydro-betanin (neobetanin) and their decarboxylated derivatives while mixture 2 consisted of decarboxy- and dehydro-betacyanins. The products of mixture 1 arose during thermal degradation of betanin/isobetanin in mild conditions while the dehydro-betacyanins of mixture 2 appeared after longer heating of the juice from B. vulgaris L. Two solvent systems were found to be effective for the HPCCC. A highly polar, high salt concentration system of 1-PrOH–ACN–(NH4)2SO4 (satd. soln)–water (v/v/v/v, 1:0.5:1.2:1) (tail-to-head mode) enabled the purification of 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (all from mixture 1) plus 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2-decarboxy-neobetanin and 2,15,17-tridecarboxy-neobetanin (from mixture 2). The other solvent system included heptafluorobutyric acid (HFBA) as ion-pair reagent and consisted of tert-butyl methyl ether (TBME)–1-BuOH–ACN–water (acidified with 0.7% HFBA) (2:2:1:5, v/v/v/v) (head-to-tail mode). This system enabled the HPCCC purification of 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (from mixture 1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin and 2,15,17-tridecarboxy-neobetanin (mixture 2). The results of this research are crucial in finding effective isolation methods of betacyanins and their derivatives which are meaningful compounds due their colorant properties and potential health benefits regarding antioxidant and cancer prevention. The pigments were detected by LC-DAD and LC–MS/MS techniques.
Keywords: Betanin; Betalains; Betacyanins; Counter-current chromatography; Beta vulgaris L;

A novel sensitive method based on tertiary amine labeling for the analysis of gibberellins (GAs) by capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection was proposed. GA3 was tagged with 2-(2-aminoethyl)-1-methylpyrrolidine (AEMP) using N, N′-dicyclohexylcarbodiimide (DCC) and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) as coupling agents in acetonitrile to produce GA3-AEMP-derivative. The GA3-AEMP-derivative was injected into CE by electrokinetic injection and detected by Ru(bpy)3 2+-based ECL. The parameters affecting derivatization, detection and separation such as concentration of reactants, detection potential, pH and concentration of separation buffer, were investigated in detail. Under optimum conditions, the linear concentration range for GA3 was from 2.0 × 10−7 to 1.28 × 10−4  M with a correlation coefficient of 0.9997. The detection limit was 8 × 10−8  M (S/N = 3). The relative standard deviations of migration time, peak intensity and peak area for nine continuous injections of 2.0 × 10−5  M GA3-AEMP-derivative were 1.0%, 2.1% and 4.2%, respectively. The developed approach was successfully applied to the determination of total GAs in the stem, leaf and seed of soybean (Glycine max [L.] Merr.) with recoveries in the range from 89.6% to 99.3%.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Gibberellin A3; Soybean; Tertiary amine labeling;

A generic screening methodology for horse doping control by LC–TOF-MS, GC–HRMS and GC–MS by Maroula K. Kioussi; Emmanouil M. Lyris; Yiannis S. Angelis; Maria Tsivou; Michael A. Koupparis; Costas G. Georgakopoulos (69-80).
In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17β-sulfate steroid conjugates. The extracts were treated for LC–TOF-MS, GC–HRMS and GC–MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC–TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC–MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference – specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples.
Keywords: Screening; Equine; Urine; Doping control; Mass spectrometry; Spectra deconvolution;

Analysis of free, mono- and diacetylated polyamines from human urine by LC–MS/MS by Merja R. Häkkinen; Antti Roine; Seppo Auriola; Antti Tuokko; Erik Veskimäe; Tuomo A. Keinänen; Terho Lehtimäki; Niku Oksala; Jouko Vepsäläinen (81-89).
Polyamines are promising biochemical markers of cancer and many other pathophysiological conditions, and thus their concentrations in biological fluids are a matter of interest. However, since the concentrations of these compounds are low, their quantitation is typically based on methods requiring laborious sample preparation. Here we developed and validated an LC–MS/MS method to analyze simultaneously free (DAP, PUT, CAD, SPD, SPM) monoacetylated (AcPUT, AcCAD, N 1AcSPD, N 8AcSPD, N 1AcSPM) and diacetylated (DiAcPUT, DiAcCAD, DiAcSPD, DiAcSPM) polyamines from human urine without the need for derivatization. Deuterium labeled polyamines were the internal standards for each analyte. Diluted urine samples spiked with internal standards were filtered through a strong anion exchange resin prior to LC–MS/MS analysis. The chromatographic separation of 14 polyamines was achieved in 12 min on C18 column with 0.1% HFBA (v/v) as the ion-pairing agent and a water–acetonitrile gradient. Ionization was performed with positive electrospray ionization (ESI) and detection was with a triple quadrupole mass spectrometer with selected reaction monitoring. Calibration curves ranged from up to 5 to 10,000 nM. The accuracy and precision of the method were determined using urine based quality control samples, and matrix effects were examined by using standard addition methods. This novel method is suitable for elucidating differences in urinary polyamine excretion in cancer patients and healthy humans.
Keywords: Polyamines; N-acetylated; Urine; Liquid chromatography–tandem mass spectrometry; Cancer diagnostic markers; Prostate cancer;

Metabolite identification of crude extract from Ganoderma lucidum in rats using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry by Chun-Ru Cheng; Min Yang; Kate Yu; Shu-Hong Guan; Xiao-Hui Wu; Wan-Ying Wu; Yan Sun; Chuan Li; Jie Ding; De-An Guo (90-99).
The metabolism of traditional Chinese medicine is very complicated and has been a great challenge. In the present paper, a new strategy was established to study the metabolism of crude extract from Ganoderma lucidum using the highly separative and sensitive ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Based on the investigation of the metabolism of five representative single compounds (ganoderic acid), a total of 90 metabolites were identified from the bile sample after oral administration of the crude extract. Among them, 21 compounds were identified by comparison with the reference standards, the other unknown metabolites were tentatively characterized by interpretation of the high resolution low collision energy and high collision energy mass spectra using the fragmentation rules. The metabolic characteristics and “soft spots” of the ganoderic acids were revealed. After being absorbed, the ganoderic acids from the extract could undergo extensive phases I and II metabolism in rat before excreted into the bile. The main ganoderic acids could transform from one to another through reduction, oxidation, deacetylation and desaturation reactions. Other metabolic transformation included hydroxylation, sulfation and glucuronidation. The total tendency was that the low polar ganoderic acids were transformed into the high polar metabolites to eliminate from the organism. The metabolic “soft spots” of the ganoderic acids were 3,7,15,23-carbonyl groups (or hydroxyl groups), angular methyl groups, 20(22)-double bond, 12-acetoxyl group and 26-carboxylic acid moiety. These results are considered to be important for the further investigation of G. lucidum.
Keywords: Ganoderma lucidum; Ganoderic acid; Metabolism; Metabolite identification; UPLC/Q-TOF;

The development and validation of a LC–MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC–MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29–70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16–56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5–5000 ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100 ± 10%), and precision (<11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC–MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation.
Keywords: Lapatinib; High performance liquid chromatography (HPLC); Mass spectrometry; LC–MS/MS; Recovery; Internal standard;

Quantitation of neonicotinoid metabolites in human urine using GC-MS by Hiroshi Nomura; Jun Ueyama; Takaaki Kondo; Isao Saito; Katsuyuki Murata; Toyoto Iwata; Shinya Wakusawa; Michihiro Kamijima (109-115).
A rapid and sensitive analytical method using gas chromatography-mass spectrometry (GC-MS) was developed for the measurement of neonicotinoid (NEO) metabolites 6-chloronicotinic acid (6CN), 2-chloro-1,3-thiazole-5-carboxylic acid (2CTCA) and 3-furoic acid (3FA) from human urine. After acid hydrolysis, the metabolites were extracted using solid phase extraction (SPE) column (Bond Elute Plexa PCX) and eluted with methanol. N,O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA-TMCS, 99:1) was used for the derivatization of metabolites and analyzed by GC-MS with the electron ionization mode. The elution solvent, derivatization reagent and its conditions were mainly optimized for improved detection and quantitation of the metabolites based on signal-to-noise ratio, recoveries and reproducibility. Our present method offered a sufficiently low limit of detection (0.1 μg/L for each metabolite) with satisfactory within-run and between-day accuracy and precision (variability less than 12.3%, R.S.D). This method is simple, sensitive and precise, and has been successfully applied to quantify low concentrations of urinary 6CN, 2CTCA and 3FA for the occupational NEO exposures survey
Keywords: Neonicotinoid; Metabolite; GC-MS; Urine;

Liquid chromatographic–mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria by Natthida Sriboonvorakul; Natchanun Leepipatpiboon; Arjen M. Dondorp; Thomas Pouplin; Nicholas J. White; Joel Tarning; Niklas Lindegardh (116-122).
Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5–2500 μg/mL for LA, 0.125–125 μg/mL for aHBA, 7.5–375 μg/mL for bHBA, 0.1–100 μg/mL for pHPLA, 1–1000 μg/mL for MA, 0.25–250 μg/mL for MMA, 0.25–100 μg/mL for EMA, and 30–1500 μg/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA = 177–1169 μg/mL, aHBA = 4.70–38.4 μg/mL, bHBA = 7.70–38.0 μg/mL, pHPLA = 0.900–4.30 μg/mL and aKGA = 30.2–32.0) and seven in urine samples (range LA = 11.2–513 μg/mL, aHBA = 1.50–69.5 μg/mL, bHBA = 8.10–111 μg/mL, pHPLA = 4.30–27.7 μg/mL, MMA = 0.300–13.3 μg/mL, EMA = 0.300–48.1 μg/mL and aKGA = 30.4–107 μg/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.
Keywords: Acidosis; Severe malaria; Liquid chromatography; Mass spectrometry; Bio-analysis; Unidentified acids;

A three phase hollow fiber liquid-phase microextraction with in situ derivatization (in situ HF-LPME) followed by high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed for the trace determination of metformin hydrochloride (MH) in biological fluids. A new derivatization agent pentafluorobenzoyl chloride (PFBC) was used. Several parameters that affect the derivatization and extraction efficiency were studied and optimized (i.e., type of organic solvent, volume of NaOH (4 M) and derivatization agent in the donor phase, acceptor phase (HCl) concentration, stirring speed, temperature, time and salt addition). Under the optimum conditions (organic solvent, dihexyl ether; volume of NaOH (4 M) and derivatization agent (10 mg PFBC in 1 mL acetonitrile) in the donor phase, 600 and100 μL, respectively; acceptor phase, 100 mM HCl (10 μL); stirring speed, 300 rpm; extraction time, 30 min; derivatization temperature, 70 °C; without addition of salt) an enrichment factor of 210-fold was achieved. Good linearity was observed over the range of 1–1000 ng mL−1 (r 2  = 0.9998). The limits of detection and quantitation were 0.56 and 1.68 ng mL−1, respectively. The proposed method has been applied for the determination of MH in biological fluids (plasma and urine) and water samples. Prior to the microextraction treatment of plasma samples, deproteinization step using acetonitrile was conducted. The proposed method is simple, rapid, sensitive and suitable for the determination of MH in a variety of samples.
Keywords: HF-LPME; HPLC-UV; Anti-diabetic; Metformin hydrochloride; Biological fluids;