Journal of Chromatography B (v.944, #C)

Quantification of peramivir in dog plasma by liquid chromatography/tandem mass spectrometry employing precolumn derivatization by Xin Li; Ying Li; Juan Wang; Lili Wang; Wu Zhong; Jinxiu Ruan; Zhenqing Zhang (1-5).
Peramivir is a novel influenza neuraminidase inhibitor used for anti-influenza. In this article, a novel method was developed to determine peramivir in dog plasma using a derivatization treatment step to increase the retention time and enhance the signal intensity. The sample preparation consisted of a protein precipitation extraction followed by derivatization with 10 M hydrochloric acid–methanol (10:90, v/v) and determined by liquid chromatography coupled with tandem mass spectrometry. The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 343 → 284 and m/z 299 → 152 were used to measure the derivative of peramivir and Ro 64-0802 (internal standard, an active metabolite of oseltamivir). The chromatographic separation was achieved using a ZORBAX RX-C8 (2.0 mm × 150 mm × 5 μm) analytical column with an isocratic mobile phase composed of acetonitrile–water–formic acid (30:70:0.1, v/v/v, 0.2 mL/min). The method was linear over a concentration range of 0.25–250 ng/mL. The average intra-day/inter-day precision values were 4.04–8.17% and 3.02–7.08%, respectively, while the average accuracy value was 93.99–106.48%. This method has been successfully applied to the preclinical dog research of peramivir following intragastric administration.
Keywords: Peramivir; Influenza virus neuraminidase inhibitor; Derivatization; Liquid chromatography–tandem mass spectrometric method (LC–MS/MS); Dog plasma;

A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of YZG-331 in mouse plasma was developed. Plasma samples containing YZG-331 and YZG-441 (internal standard, IS) were prepared using a simple protein precipitation by the addition of acetonitrile. Thermo Scientific TSQ Quantum triple quadrupole system with multiple reaction monitoring (MRM) positive scanning mode was applied. The separation was performed on a ZorbaxSB-C18 column (3.5 μm, 2.1 mm × 100 mm) at a flow rate of 0.2 mL/min, using acetonitrile/water containing 0.1% formic acid (v/v) as mobile phase. The MS/MS ion transit ions monitored were 386→254 for YZG-331 and 400→268 for IS. Linear detection responses were obtained for YZG-331 ranging from 25 to 5000 ng/mL and the lower limits of quantitation (LLOQ) for the compound was 25 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 12.6% for all analytes, while the deviation of assay accuracies was within ±6.9%. The average recoveries of analytes were greater than 94.3%. 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 YZG-331 in mice.
Keywords: YZG-331; LC–MS/MS; Mouse; Pharmacokinetic;

24-Dehydropollinstanol (DEH), 24-methylene cholesterol (MET) and 31-norcycloartenol (NOR) are the functional triterpene alcohols of pollen of Brassica campestris. To study the pharmacokinetics of the above components of pollen of B. campestris in rats, a liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed. To avoid the interference of endogenous MET in rat plasma, fetal bovine serum (FBS) was selected as surrogate matrix and validated. Rat plasma was liquid-liquid extracted, then the chromatographic separation was conducted on a poroshell 120 SB C18 column (2.7 μm, 2.1 mm × 50 mm) at 38 °C within 5.6 min utilizing a gradient elution with a mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive atmospheric pressure chemical ionization (APCI). The method was validated over the concentration of 9.8–1560 ng/ml; the inter-and-intra-day precisions (RSD %) were ≤7.8%, and the accuracies (RE %) were −5.3% to 12.2%, the extraction recovery ranged from 73.5% to 106.9% for all of these analytes, and no obvious matrix effect was observed. The developed method was applied successfully to study the pharmacokinetics of DEH, MET and NOR in rats after oral administration of pollen of B. campestris.
Keywords: 24-Dehydropollinstanol; 24-Methylene cholesterol; 31-Norcycloartenol; Pharmacokinetics; Pollen of Brassica campestris; LC–MS/MS;

Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry by Leah C. Jimmerson; Jia-Hua Zheng; Lane R. Bushman; Christine E. MacBrayne; Peter L. Anderson; Jennifer J. Kiser (18-24).
Efficient, inexpensive and sensitive assays for the measurement of drugs are of interest for pharmacokinetic and pharmacodynamics (PK–PD) analysis. Dried blood spots (DBS) are a unique bioanaltyical matrix with the potential to fulfill this interest for the measurement of numerous analytes. Here we describe the development and validation of a reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of ribavirin (RBV) in DBS. A 3 mm punch from spotted and dried whole blood was extracted in methanol utilizing isotopically labeled internal standard for LC–MS/MS analysis. Validation was performed over a range of 0.05 μg/mL to 10.0 μg/mL and the method was shown to be precise (coefficient of variation ≤15%) and accurate (within ±15% of control). These acceptance criteria were met for hematocrit ranges of 20–54%, for center versus edge punches and for spot volumes from 10 to 60 μL. RBV was stable for up to 140 days at room temperature and −20 °C as well as for three freeze/thaw cycles. Correlation of RBV in DBS versus in plasma yielded r 2  ≥ 0.98 demonstrating that DBS can be used as an alternative to plasma for PK–PD studies in human subjects.
Keywords: Ribavirin; Dried blood spot; Analytical method; Nucleoside analog; Hepatitis C; LC–MS/MS;

Determination of carbadox and olaquindox metabolites in swine muscle by liquid chromatography/mass spectrometry by Tomasz Sniegocki; Malgorzata Gbylik-Sikorska; Andrzej Posyniak; Jan Zmudzki (25-29).
This paper presents LC–MS/MS method that was developed for the simultaneous determination and confirmation metabolites of carbadox (desoxycarbadox, quinoxaline-2-carboxylic) and olaquindox (3-methylquinoxaline-2-carboxylic acid) residues in pig muscle tissues at concentrations ≤3.0 μg kg−1. Pig muscle tissues were deproteinated with meta-phosphoric acid in methanol and then were extracted with ethyl acetate:dichloromethane (50:50, v/v). The whole extracts were evaporated to dryness in rotary evaporator at 45 °C, and dry residues were re-dissolved in 0.5% isopropanol in 1% acetic acid. The LC separation was performed on a C8 column with a gradient system consisting of isopropanol/water/acetic acid and methanol as the mobile phase. Additionally SelexION™ technology to reduce matrix effect was used. The decision limit (CCα) ranged from 1.04 μg kg−1 to 2.11 μg kg−1 and the detection capability (CCβ) ranged from 1.46 μg kg−1 to 2.89 μg kg−1. The total recoveries were from 99.8% to 101.2%. The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC.
Keywords: Carbadox; Olaquindox; Metabolites; SelexION™;

Purification of antibiotics from the biocontrol agent Streptomyces anulatus S37 by centrifugal partition chromatography by Olivier Couillerot; Souad Loqman; Alix Toribio; Jane Hubert; Léa Gandner; Jean-Marc Nuzillard; Yedir Ouhdouch; Christophe Clément; Essaid Ait Barka; Jean-Hugues Renault (30-34).
A novel actinomycete strain, Streptomyces anulatus S37, has been isolated from the rhizosphere of healthy Moroccan Vitis vinifera on the basis on its ability to promote grapevine growth and to induce natural defences against various phytopathogens. In the present work, the main bioactive metabolites produced by S. anulatus S37 were isolated. A crude n-BuOH extract of the S37 fermentation broth was firstly partitioned in a biphasic solvent system composed of n-heptane, methanol, and water (5:1.5:3.5, v/v). The most active organic fraction (1.1 g) as revealed by TLC-bioautography was subsequently separated by a two-step centrifugal partition chromatography procedure. The first separation was performed in the ascending mode at 6 mL/min with the biphasic solvent system n-heptane, ethyl acetate, methanol and water (2:1:2:1, v/v), to finally recover 40 mg of a pure compound identified as streptochlorin by NMR spectroscopy. In a second separation, the solvent system n-heptane, acetonitrile, and water (5:5:4, v/v) was used in the ascending mode at 3 mL/min to purify 135 mg of nigericin and 53 mg of piericidin A1. Assays performed with the three compounds have confirmed their inhibitory impact on the growth of Botryris cinerea in dual confrontation and also on V. vinifera L. plantlets.
Keywords: Streptomyces; Centrifugal partition chromatography; Botrytis cinerea; Vitis vinifera;

Determination of sec-O-glucosylhamaudol in rat plasma by gradient elution liquid chromatography–mass spectrometry by Congcong Wen; Chongliang Lin; Xiaojun Cai; Jianshe Ma; Xianqin Wang (35-38).
Sec-O-glucosylhamaudol is one of the major bioactive compounds of the Saposhnikoviae Radix. A simple and selective liquid chromatography–mass spectrometry (LC–MS) method for determination of sec-O-glucosylhamaudol in rat plasma was developed. After addition of carbamazepine as internal standard (IS), protein precipitation with acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 439 for sec-O-glucosylhamaudol and m/z 237 for the IS. Calibration plots were linear over the range of 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma. Mean recovery of sec-O-glucosylhamaudol in plasma was in the range of 74.8–83.7%. Intra-day and inter-day precision were both <15%. This method was successfully applied in pharmacokinetic study after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol in rats.
Keywords: Sec-O-glucosylhamaudol; LC–MS; Pharmacokinetics; Rat plasma;

Negative pseudo-affinity chromatography for plasmid DNA purification using berenil as ligand by C. Caramelo-Nunes; M.F. Gabriel; P. Almeida; J.C. Marcos; C.T. Tomaz (39-42).
The present study, reports the utilization of berenil as ligand in a negative pseudo-affinity chromatographic step to purify the plasmid pVAX1-LacZ from Escherichia coli clarified lysates. The chromatographic support was prepared by coupling berenil to epoxy-activated Sepharose and was qualitatively and quantitatively characterized using scanning electron microscopy, Fourier transformed infrared spectroscopy and elemental analysis. The clarified lysate was loaded onto the berenil-Sepharose support with 0.55 M of ammonium sulphate in the eluent, achieving the immediate elution of plasmid DNA. The impurities tightly bound to the support, were eluted after decreasing the salt concentration to 0 M. The overall process enabled the recovery of 87% of loaded plasmid DNA with a HPLC purity of ≫99% and according to FDA specifications. This method represents an alternative approach to the previous utilization of the same chromatographic pseudo-affinity support in a positive mode. It uses lower amounts of salt and one-step chromatographic procedure, resulting in smaller operating time and costs and representing an alternate procedure for plasmid DNA purification.
Keywords: Berenil-Sepharose; Negative pseudo-affinity chromatography; Plasmid purification; Small DNA ligands;

A simple, selective, and sensitive high performance liquid chromatography (HPLC) procedure has been developed for determination of trazodone in human plasma. Prazosin was employed as the internal standard (IS). Sample preparation involved liquid–liquid extraction by methyl tert-butyl ether after alkalinization with ammonia. The HPLC separation was performed on a CAPCELL PAK SCX column (250 mm × 4.6 mm, 5.0 μm, Shiseido, Japan) with a mobile phase of acetonitrile/80 mmol/L ammonium phosphate (pH adjusted to 6.0) (60:40, v/v) at a flow rate of 1.2 mL/min. The peaks were detected by using fluorescence detector (excitation wavelength 320 nm and emission wavelength 440 nm). The extraction recovery was 72.6–88.3% and the method was over the concentration range of 5.0–2486 ng/mL with a lower limit of quantitation (LLOQ) of 5.0 ng/mL using 300 μL of plasma. The intra- and inter-day accuracy of the method at three concentrations ranged from 96.7% to 104.2% for trazodone with precision of 2.9–3.7%. This validated method was successfully applied to a pharmacokinetic study enrolling 12 Chinese volunteers administered a single oral trazodone hydrochloride extended-release tablet of 75 mg.
Keywords: Trazodone; Fluorescence detector; Strong cation exchange column; Human plasma; Pharmacokinetics;

Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples by Beauty Arora; Angayarkanni Narayanasamy; Jayabalan Nirmal; Nabanita Halder; Santosh Patnaik; Alok K. Ravi; Thirumurthy Velpandian (49-54).
The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC–MS/MS. The gradient elution of HCTL was achieved within 5 min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5–32.5 nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at −86 °C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.
Keywords: Homocysteine thiolactone; LC–MS/MS; HILIC; Plasma; Validation; Stability;

Adduct formation in liquid chromatography-triple quadrupole mass spectrometric measurement of bryostatin 1 by Thomas J. Nelson; Abhik Sen; Daniel L. Alkon; Miao-Kun Sun (55-62).
Bryostatin 1, a potential anti-Alzheimer drug, is effective at subnanomolar concentrations. Measurement is complicated by the formation of low m/z degradation products and the formation of adducts with various cations, which make accurate quantitation difficult. Adduct formation caused the sample matrix or mobile phase to partition bryostatin 1 into products of different mass. Degradation of the 927 [M+Na]+ ion to a 869  m/z product was strongly influenced by ionization conditions. We validated a bryostatin 1 assay in biological tissues using capillary column HPLC with nanospray ionization (NSI) in a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode. Adduct formation was controlled by adding 1 mM acetic acid and 0.1 mM sodium acetate to the HPLC buffer, maximizing the formation of the [M+Na]+ ion. Efficient removal of contaminating cholesterol from the sample during solvent extraction was also critical. The increased sensitivity provided by NSI and capillary-bore columns and the elimination of signal partitioning due to adduct formation and degradation in the ionization source enabled a detection limit of 1 × 10−18  mol of bryostatin 1 and a LLOQ of 3 × 10−18  mol from 1 μl of sample. Bryostatin 1 at low pmol/l concentrations enabled measurement in brain and other tissues without the use of radioactive labels. Despite bryostatin 1's high molecular weight, considerable brain access was observed, with peak brain concentrations exceeding 8% of the peak blood plasma concentrations. Bryostatin 1 readily crosses the blood–brain barrier, reaching peak concentrations of 0.2 nM, and specifically activates and translocates brain PKCɛ.
Keywords: Mass spectrometry; Bryostatin 1; Paclitaxel; Adduct; Alzheimer's disease; LC–MS;

Determination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies by Osama Y. Alshogran; Andrew J. Ocque; Jielu Zhao; Billy W. Day; François A. Leblond; Vincent Pichette; Thomas D. Nolin (63-68).
A sensitive, accurate, and reproducible ultra-high performance liquid chromatography–tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10 mM Tris–HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid–liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1 mm × 100 mm, 1.9 μm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4 mL/min and the total run time was 5 min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100–10,000 ng/mL for warfarin, and 0.5–250 ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.
Keywords: Warfarin alcohols; Warfarin; Reduction; LC–MS; Enzyme kinetics;

Determination of bile acids by hollow fibre liquid-phase microextraction coupled with gas chromatography by T. Ghaffarzadegan; M. Nyman; J.Å. Jönsson; M. Sandahl (69-74).
A method based on hollow-fibre liquid phase microextraction combined with gas chromatography was developed for determination of specific bile acids in caecal materials of rats. Nine unconjugated bile acids, including the primary bile acids (cholic acid, chenodeoxycholic acid and α-muricholic acid) and the secondary bile acids (lithocholic acid, deoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, β-muricholic acid and ω-muricholic acid) were quantified. Extraction conditions were evaluated, including: sample pH, type of organic solvent and amount of caecal material to be extracted. To compensate for sample matrix effects during extraction the method of standard addition was applied. The satisfactory linearity (r 2  > 0.9840), high recovery (84.2–108.7%) and good intra-assay (6.3–10.6%) and inter-assay (6.9–11.1%) precision illustrated the good performance of the present method. The method is rapid, simple and capable of detecting and determining bile acids with limit of detection (LOD) ranged from 0.002 to 0.067 μg/mL and limits of quantification (LOQ) varied from 0.006 to 0.224 μg/mL. The results indicated that the concentration of some secondary bile acids, which usually are associated with health problems, were lower in rats fed with fermentable dietary fibre compared with a fibre free control diet, while the concentration of primary bile acids, usually connected with positive health effects, were higher in rats fed with diets containing dietary fibre. Of the dietary fibres, guar gum and to some extent the mixture of pectin + guar gum had the most positive effects. Thus, it was concluded that the composition of bile acids can be affected by the type of diet.
Keywords: Bile acids; Hollow fibre liquid-phase microextraction; Caecum; Gas chromatography;

We report an accurate quantification of free sialic acid (SA) in human plasma using LC–MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC–MS/MS analysis of free SA. In addition, the use of 13C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20 ng/mL and a total running time of 4.2 min, which is the most sensitive and quick measurement for free SA in biomatrices.
Keywords: Free sialic acid; Human plasma; Isotope-labeled standard calibration; Liquid chromatography–tandem mass spectrometry; Quinoxaline derivatization;

A rapid and sensitive ultra fast performance liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of five bioactive secolignans in Peperomia dindygulensis extract, including peperomin A, peperomin B, peperomin C, 4″-hydroxypeperomin B and 4″-hydroxypeperomin C in rat plasma. Arctigenin was used as the internal standard. The separation was performed on an Innovation™ Polar-RP C18 column by a gradient elution within a runtime of 7 min. The mobile phase consisted of A (methanol) and B (0.1% formic acid in water) at a flow rate of 0.4 mL/min. The detection was accomplished by using positive ion TurboIonSpray ionization in multiple reaction monitoring mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9972. The lower limits of quantification were 1.1 ng/mL for peperomin A, 1.24 ng/mL for peperomin B, 1.02 ng/mL for peperomin C, 1.91 ng/mL for 4″-hydroxypeperomin B and 1.27 ng/mL for 4″-hydroxypeperomin C. The intra- and inter-day precision (RSD%) was within 15% and the accuracy (RE%) ranged from −11.7% to 10.3%. This simple and sensitive method was fully validated and successfully applied to the pharmacokinetic study of peperomin A, peperomin B, peperomin C, 4″-hydroxypeperomin B and 4″-hydroxypeperomin C in rat plasma after oral administration of P. dindygulensis extract.
Keywords: Peperomia dindygulensis; Secolignans; Rat plasma; Pharmacokinetics; UFLC–MS/MS;

A sensitive UPLC–MS/MS method for simultaneous determination of eleven bioactive components of Tong-Xie-Yao-Fang decoction in rat biological matrices by Tian-xue Li; Lang Hu; Meng-meng Zhang; Jian Sun; Yue Qiu; Jun-qian Rui; Xing-hao Yang (90-100).
There is a growing concern for the sensitive quantification of multiple components using advanced data acquisition method in herbal medicines (HMs). An improved and rugged UPLC–MS/MS method has been developed and validated for sensitive and rapid determination of multiply analytes from Tong-Xie-Yao-Fang (TXYF) decoction in three biological matrices (plasma/brain tissue/urine) using geniposide and formononetin as internal standards. After solid-phase extraction, chromatographic separation was performed on a C18 column using gradient elution. Quantifier and qualifier transitions were monitored using novel Triggered Dynamic multiple reaction monitoring (TdMRM) in the positive ionization mode. A significant peak symmetry and sensitivity improvement in the TdMRM mode was achieved as compared to conventional MRM. The reproducibility (RSD%) was ≤7.9% by applying TdMRM transition while the values were 6.8–20.6% for MRM. Excellent linear calibration curves were obtained under TdMRM transitions over the tested concentration ranges. Intra- and inter-day precisions (RSD%) were ≤14.2% and accuracies (RE%) ranged from −9.6% to 10.6%. The validation data of specificity, carryover, recovery, matrix effect and stability were within the required limits. The method was effectively applied to simultaneously detect and quantify 1 lactone, 2 monoterpene glucosides, 1 alkaloid, 5 flavonoids and 2 chromones in plasma, brain tissue and urine after oral administration of TXYF decoction. In conclusion, this new and reliable method is beneficial for quantification and confirmation assays of multiply components in complex biological samples.
Keywords: Tong-Xie-Yao-Fang; UPLC–MS/MS; Triggered Dynamic multiple reaction monitoring; Biological matrices; Bio-analysis; Multi-component determination;

A specific and reliable HPLC–MS/MS method was developed and validated for the simultaneous determination of six alkaloids in rat plasma, jatrorrhizine, berberine, tetrahydropalmatine, protopine, bicuculline and palmatine. The analytes were separated on a C18 column (50 mm × 2.1 mm, 1.8 μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The plasma sample was prepared by the simple protein precipitation and the recovery for the six analytes was over 80%. The calibration curves were linear over a concentration range of 0.38–1900.0 ng/mL for jatrorrhizine, 0.57–2850.0 ng/mL for berberine, 0.32–1600.0 ng/mL for tetrahydropalmatine, 0.21–1050.0 ng/mL for protopine, 0.34–1700.0 ng/mL for bicuculline and 0.22–1100 ng/mL for palmatine. The intra-day and inter-day precision was less than 15% and the relative error (RE) was all within ±15%. The validated method was successfully applied to a pharmacokinetics study in rats after oral administration of the extracts of Rhizoma Corydalis Decumbentis (a famous Chinese herb).
Keywords: HPLC–MS/MS; Alkaloid; Rhizoma Corydalis Decumbentis; Pharmacokinetics; Rat plasma;

Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2 mL, 10.7 mg capacity) and piscine heparin (5 mL, 2.7 mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16 cm × 16 cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1 mg anti-human fibrinogen in 8.4 mL resin) and serum albumin (0.42 mg binding capacity in 14 mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography–mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50 cm × 75 μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome.
Keywords: Affinity chromatography; Fibrinogen; Heparin-binding; Biomarkers; Immunodepletion;

Method development and validation for simultaneous determination of lumefantrine and its major metabolite, desbutyl lumefantrine in human plasma using RP-HPLC/UV detection by Fazli Khuda; Zafar Iqbal; Yasar Shah; Lateef Ahmmad; Fazli Nasir; Amir Zada Khan; Amanullah; Naila Shahbaz (114-122).
A simple, specific, precise and rapid RP-HPLC–UV method was developed for simultaneous determination of lumefantrine and its metabolite desbutyl lumefantrine in human plasma. Experimental parameters were optimized and the method was validated according to standard guidelines. The method showed adequate separation for lumefantrine and desbutyl lumefantrine and best resolution was achieved with Supelco Discovery HS C18 RP (150 mm × 4.6 mm, 5 μm) column using acetonitrile and 0.05% trifluroacetic acid (70:30, v/v) as a mobile phase pumped at a flow rate of 1.0 ml/min and wavelength of 335 nm. The method was linear over the concentration range of 10–12,000 ng/ml. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for lumefantrine were 10.0 and 18.0 ng/ml, while for desbutyl lumefantrine were 7.5 and 15.0 ng/ml, respectively. The proposed method was efficiently applied for determination of lumefantrine and desbutyl lumefantrine concentrations in plasma samples for pharmacokinetic studies.
Keywords: Lumefantrine; Desbutyl lumefantrine; RP-HPLC/UV; Validation;

An ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with MetaboLynx™ software combined with mass defect filtering (MDF) method were provided for orientin metabolism study. The chromatographic separation was performed on a 1.7 μm particle size Syncronis C18 column using gradient elution system. The components were identified and confirmed according to the mass spectrometric fragmentation mechanisms, MS/MS fragment ions and relevant literature by means of electrospray ionization mass spectrometry in negative ion mode. With this method, a total of three metabolites were identified based on retention time and MS/MS data. The results illustrated that deglycosylation, dehydroxylation and acetylation were the major metabolic pathways of orientin in vitro by human intestinal bacteria. Additionally, colonic bacteria were screened for bacteria involved in the conversion of orientin. A gram-negative anaerobic bacterium, strain 45, was newly isolated from healthy human feces. This strain, which was able to cleave the C-glycoside of orientin to produce luteolin and generate some other metabolites, had the similarity of 95.44% with Enterococcus casseliflavus and was named Enterococcus sp. 45 based on 16S rRNA sequence analysis. In this paper, the metabolic routes, metabolites of oreintin produced by the intestinal bacteria and the Enterococcus sp. 45 were investigated for the first time.
Keywords: Enterococcus sp. 45; Metabolites; Orientin; 16s rRNA; UPLC-Q-TOF/MS;

Structural elucidation of the metabolites of lapachol in rats by liquid chromatography–tandem mass spectrometry by Lu Bai; Ying Han; Jinfeng Yao; Xiaorong Li; Yuhang Li; Pinxiang Xu; Ming Xue (128-135).
Lapachol is a natural naphthoquinone compound derived from Bignoniaceae (Tabebuia sp.) that possesses a range of significant biological activities. Nine phase I and four phase II metabolites of lapachol in rat bile were firstly elucidated and identified using a sensitive LC-ESI–MS n method. The molecular structures of the metabolites have been presented on the basis of the characteristics of their precursor and product ions, as well as their fragmentation mechanisms and chromatographic retention times. The results indicated that the phase I metabolites were predominantly biotransformed by the hydroxylation, semiquinone hydrogenation at the oxygen position or a side chain rearrangement. The phase II metabolites were identified as the glucuronidated conjugates which showed a characteristic neutral loss of 176 Da. Based on the results of this research, we have proposed the metabolic pathways for lapachol in rats. This work has provided novel information for the in vivo lapachol metabolism which could be used to develop a novel drug candidate, as well as a better understanding of the safety and efficacy of the drug.
Keywords: Structural elucidation; Lapachol; Metabolites; LC-ESI–MS n ;

Fast determination of paraquat in plasma and urine samples by solid-phase microextraction and gas chromatography–mass spectrometry by Lina Gao; Junting Liu; Chunyuan Wang; Guojie Liu; Xiaodong Niu; Cuixia Shu; Juan Zhu (136-140).
A simple, sensitive and reliable gas chromatographic–mass spectrometric method (GC–MS) for quantifying paraquat concentration in biological samples has been developed, using ethyl paraquat as an internal standard. The method involved the procedures of sodium borohydride–nickel chloride (NaBH4–NiCl2) reduction and solid-phase microextraction (SPME) of the perhydrogenated products. GC–MS was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. Under the optimal conditions, recoveries in plasma and urine samples were 94.00–99.85% and 95.00–100.34%, respectively. Excellent sample clean-up was observed and good linearities (r  = 0.9982 for plasma sample and 0.9987 for urine sample) were obtained in the range of 0.1–50 μg/mL. The limits of detection (S/N = 3) were 0.01 μg/mL in plasma and urine samples. The intra-day precision was less than 8.43%, 4.19% (n  = 3), and inter-day precision was less than 10.90%, 10.49% (n  = 5) for plasma and urine samples, respectively. This method was successfully applied to the analysis of the biological samples collected from a victim who died as a result of ingestion of paraquat.
Keywords: Paraquat; Plasma and urine samples; SPME; GC–MS;

Determination of urinary alpha-aminoadipic semialdehyde by LC–MS/MS in patients with congenital metabolic diseases by Isaac Ferrer-López; Pedro Ruiz-Sala; Begoña Merinero; Celia Pérez-Cerdá; Magdalena Ugarte (141-143).
This paper describes a full detailed high performance liquid chromatography/tandem mass spectrometry method for the identification and quantification of human urine alpha-aminoadipic semialdehyde, biomarker of pyridoxine-dependent epilepsy. The ionization mode of the electrospray interface was negative and the metabolite was detected in the multiple reaction monitoring mode. Intra-day and inter-day laboratory precision were 4.64% and 7.30%, respectively, total run time was 3.5 min. The calibration curve was linear between 0.25 and 10 nmol with a correlation coefficient of the calibration line (R 2  ≥ 0.9984); the limit of quantification was 0.25 nmol within the control group. This simple, fast, high reproducible and robust procedure facilitates a rapid diagnosis of patients with pyridoxine-dependent epilepsy and can also be used to confirm the elevated urinary alpha-aminoadipic semialdehyde excretion in patients with other metabolic diseases as molybdenum cofactor and isolated sulphite oxidase deficiencies.
Keywords: Alpha-aminoadipic semialdehyde; Pyridoxine-dependent epilepsy; Antiquitin; Molybdenum cofactor deficiency; Isolated sulphite oxidase deficiency;

HPLC separation of human serum albumin isoforms based on their isoelectric points by Lucía Turell; Horacio Botti; Lucía Bonilla; María José Torres; Francisco Schopfer; Bruce A. Freeman; Larissa Armas; Alejandro Ricciardi; Beatriz Alvarez; Rafael Radi (144-151).
Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA–SHg+), HSA with Cys34 oxidized to sulfenic acid (HSA–SOH) and HSA oxidized to sulfinate anion (HSA–SO2 ) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3–585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA
Keywords: Chromatofocusing; Human serum albumin; Redox isoforms; Plasma; Posttranslational modifications;

Human exposure to bisphenol A (BPA) is widespread. However, in recent years, bisphenol analogs such as bisphenol S (BPS) and bisphenol F (BPF) are replacing BPA in the production of some consumer products. Because human exposure to these alternative bisphenols may occur, biomonitoring of these bisphenol analogs is warranted. In the present study, we developed and validated a sensitive and selective method that uses on-line solid phase extraction coupled to high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing to measure BPA, BPF, BPS, and 11 other environmental phenols in urine. The method required a small amount of sample (100 μL) and minimal sample pretreatment. The limits of detection were 0.03 ng/mL (BPS), 0.06 ng/mL (BPF), 0.10 ng/mL (BPA), and ranged from 0.1 ng/mL to 1.0 ng/mL for the other 11 phenols. In 100 urine samples collected in 2009–2012 from a convenience group of anonymous adults in the United States, of the three bisphenols, we detected BPA at the highest frequency and median concentrations (95%, 0.72 ng/mL), followed by BPS (78%, 0.13 ng/mL) and BPF (55%, 0.08 ng/mL). This sensitive, rugged, and labor and cost-effective method could be used for the analysis of large number of samples for epidemiologic studies.
Keywords: Bisphenol A; Bisphenol F; Bisphenol S; Exposure; HPLC–MS/MS; Urine;

A simple and fast methodology to detect and identify multiple classes of lipid from human plasma is developed utilizing ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC–QTOF) as lipidomics platform. All the conditions for the sample preparation and analytical instruments were optimized in detail to detect nine lipid classes (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), triacylglyceride (TG), phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC), lysophosphatidic acid (LysoPA), and sphingomyelin (SM)), which are the most important biologically active lipids but have different characteristics. Finally, the plasma was prepared after a liquid–liquid extraction with a mixture of chloroform/methanol (1:2 v/v) including salting out by adding 0.15 M of NaCl and the residue after evaporation was reconstituted with a mixture of chloroform/methanol (1:1 v/v) to dissolve all lipids which have different polarity. The chromatographic conditions were set up such that mobile phase (A) comprised 10 mM ammonium acetate in 40% acetonitrile and mobile phase (B) comprised 10 mM ammonium acetate in acetonitrile:isopropanol = 10:90 (v/v) with ACQUITY BEH C 18 as the stationary phase. In particular, a retention time index of PC was constructed by analyzing known standards to confirm each variant of PC without the use of any additional standards in every experiment. The lipidomic methodology and the retention time index of PC were applied to analyze the lipidomic profiling of human plasma from rosuvastatin (lipid lowering drug) treated subjects.In the developed lipidomic platform, all lipids were successfully analyzed within 16 min and PCs could be confirmed with the PC retention time index. In rosuvastatin treatment, the lipid profiling was changed in all the eight lipid classes. The level of SM, TG, PI and PE decrease significantly but LysoPCs and PCs were whether decreased or increased. Those results indicated that the plasma level of overall lipids decreased by drug response, however, the changes in the lipids which are important components for biological membrane such as LysoPC and PC were more complicated, and it could be related to the side effect of rousuvastatin.In conclusion, it was found that our lipidomic methodology and the PC retention time index provided not only overall lipidomic information but also profiled specific information of drug response.
Keywords: Lipidomics; UPLC–QTOF; Lipid; Rosuvastatin; Phosphatidylcholine;

The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15 min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1 μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.
Keywords: Amino acid analysis; Underivatized; Tandem mass spectrometry;

Liquid chromatography–tandem mass spectrometry assay for therapeutic drug monitoring of the tyrosine kinase inhibitor, midostaurin, in plasma from patients with advanced systemic mastocytosis by Philippe Bourget; Alexandre Amin; Marie-Olivia Chandesris; Fabrice Vidal; Christophe Merlette; Isabelle Hirsch; Laure Cabaret; Ana Carvalhosa; Agnès Mogenet; Laurent Frenzel; Gandhi Damaj; Olivier Lortholary; Olivier Hermine (175-181).
We developed and validated quantitative bioanalytical liquid chromatography–tandem mass spectrometry assay for the protein kinase inhibitor, midostaurin. Plasma samples were pre-treated using a protein precipitation with methanol containing midostaurin-d5 as an internal standard. After centrifugation, 5 μL of the supernatant was injected into the chromatographic system. The system consisted of a 3.5 μm particle bonded octadecyl silica column, with gradient elution using a mixture of 0.1% (v/v) formic acid in acetonitrile and 10 mM ammonium formate in water with 0.1% formic acid. The analyte was quantified using the selected reaction-monitoring mode of a triple quadrupole mass spectrometer equipped with a heated electrospray interface. The assay was validated in a 75–2500 ng/mL calibration range. For quality control, within-day and between-day precisions were 1.2–2.8%, and 1.2–6.9%, respectively. The β-expectation tolerance limit (accuracy) met the limits of acceptance ±15% (±20% for the LLQ). The drug was sufficiently stable under all relevant analytical conditions. The assay has successfully been used to assess drug levels for therapeutic drug monitoring in patients presenting advanced systemic mastocytosis and treated with the promising midostaurin.
Keywords: Advanced systemic mastocytosis; LC–MS/MS; Midostaurin; Therapeutic drug monitoring; Human plasma;