Journal of Chromatography B (v.848, #2)

Methods of analysis and separation of chiral flavonoids by Jaime A. Yáñez; Preston K. Andrews; Neal M. Davies (159-181).
Although the analysis of the enantiomers and epimers of chiral flavanones has been carried out for over 20 years, there often remains a deficit within the pharmaceutical, agricultural, and medical sciences to address this issue. Hence, despite increased interest in the potential therapeutic uses, plant physiology roles, and health-benefits of chiral flavanones, the importance of stereoselectivity in agricultural, nutrition, pharmacokinetic, pharmacodynamic, pharmacological activity and disposition has often been ignored. This review presents both the general principles that allow separation of chiral flavanones, and discusses both the advantages and disadvantages of the available chromatographic assay methods and procedures used to separately quantify flavanone enantiomers and epimers in biological matrices.
Keywords: Flavonoid; Flavanone; HPLC; Chiral; Enantiomer; Epimer;

Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography by David J. Boocock; Ketan R. Patel; Guy E.S. Faust; Daniel P. Normolle; Timothy H. Marczylo; James A. Crowell; Dean E. Brenner; Tristan D. Booth; Andreas Gescher; William P. Steward (182-187).
We describe a reversed-phase HPLC method that uses gradient elution and UV detection (325 nm) to determine levels of resveratrol and identify six major conjugated metabolites in the plasma and urine of human volunteers after administration of a single oral dose of 1 g. Waters Atlantis C18 3 μm served as the stationary phase. The gradient was formed using ammonium acetate and methanol, containing 2% propan-2-ol. Detection is linear between 5 ng/mL and 500 ng/mL in plasma (5–1000 ng/mL in urine). The coefficient of variation for intra- and inter-day variation is <10%. The average recovery of resveratrol from plasma and urine is 58 ± 3%. The data presented in this report demonstrate a rapid, sensitive and accurate method for the analysis of resveratrol and its metabolites in human plasma and urine for pharmacokinetic studies.
Keywords: Resveratrol; Conjugated metabolites; HPLC; Human; Plasma; Urine;

Studies on neurosteroids XIX by Tatsuya Higashi; Akihiro Nagahama; Norihiro Otomi; Kazutake Shimada (188-199).
A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the simultaneous determination of 5α-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5α-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol–acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS–MS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization–ESI-MS–MS method are 60–150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17α,21,21,21-2H4]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 μl sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5–103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented.
Keywords: Neurosteroids; LC–ESI-MS–MS; Derivatization; Rat brain; Immobilization stress; Ethanol administration;

A capillary-based microfluidic instrument suitable for immunoaffinity chromatography by Michael C. Peoples; Terry M. Phillips; H. Thomas Karnes (200-207).
The analysis of biological samples to produce clinical or research data often requires measurement of analytes from complex biological matrices and limited volumes. Miniaturized analytical systems capable of minimal sample consumption and reduced analysis times have been employed to meet this need. The small footprint of this technology offers the potential for portability and patient point-of-care testing. A prototype microfluidic system has been developed and is presented for potential rapid assessment of clinical samples. The system has been designed for immunoaffinity chromatography as a means of separating analytes of interest from biological matrices. The instrument is capable of sub-microliter sample injection and detection of labeled antigens by long wavelength laser-induced fluorescence (LIF). The laboratory-constructed device is assembled from an array of components including two syringe pumps, a nano-gradient mixing chip, a micro-injector, a diode laser, and a separation capillary column made from a polymer/silica (PEEKsil) tube. An in-house program written with LabVIEW software controls the syringe pumps to perform step gradient elution and collects the LIF signal as a chromatogram. Initial columns were packed with silica beads to evaluate the system. Optimization of the device has been achieved by measuring flow accuracy with respect to column length and particle size. Syringe size and pressure effects have also been used to characterize the capability of the pumps. Based on test results, a 200-μm × 25-mm column packed with 1-μm silica beads was chosen for use with a 500-μL syringe. The system was tested for mixer proportioning by pumping different compositions of buffer and fluorescent dye solutions in a stepwise fashion. A linear response was achieved for increasing concentrations of fluorescent dye by online mixing (R 2  = 0.9998). The effectiveness of an acidic gradient was confirmed by monitoring pH post-column and measuring premixed solutions offline. Finally, assessment of detectability was achieved by injecting fluorescent dye solutions and measuring the signal from the LIF detector. The limit of detection for the system with these solutions was 10.0 pM or 10.0 amol on-column. As proof-of-principle, immunoaffinity chromatography was demonstrated with immobilized rabbit anti-goat IgG and a fluorescent dye-goat IgG conjugate as a model antigen.
Keywords: Microflluidics; Immunoglobulins; Immunoaffinity; Laser induced fluorescence; Capillary chromatography;

A HPLC–mass spectrometric method suitable for the therapeutic drug monitoring of everolimus by Paul J. Taylor; Michael E. Franklin; Kendon S. Graham; Peter I. Pillans (208-214).
We report here the validation of an HPLC–electrospray–tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 μl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3′-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm × 2.1 mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5 → 908.3; internal standard m/z 989.5 → 922.3). The assay was linear from 0.5 to 40 μg/l (r 2  > 0.994, n  = 11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 μg/l) were 93.4–98.2% and <10.7%, respectively (n  = 10). At the lower limit of quantification (0.5 μg/l) the inter- and intra-day analytical recovery was 94.4–95.8% with imprecision of <14.1% (n  = 10). The absolute recovery of everolimus (6.5 μg/l) and internal standard (12.5 μg/l) was 96.5 and 88.3%, respectively (n  = 3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y  = 0.973x  + 0.301 (r 2  = 0.986, n  = 71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.
Keywords: Everolimus; Transplantation; HPLC; Mass spectrometry; Therapeutic drug monitoring;

In this study, a liquid chromatography/diode array detector-atmospheric pressure chemical ionization/mass spectrometry (LC/DAD-APCI/MS) was successfully developed to identify and characterize the main flavonoids and caffeoylquinic acids (CQAs) of three common Compositae plants (Chrysanthemum morifolium Raman, Artemisia annua, and Chrysanthemum coronarium) which have been used as herbal medicine. Identifications were performed by comparing the retention time, UV and mass spectra of samples with standards or/and earlier publications. The crude methanolic extracts of these plants were assayed directly using LC/MS without any further pretreatment. The proposed method is rapid and reproducible and is useful for characterization and evaluation of different plant flavonoids and CQAs. A total of 41 different flavonoids and 6 CQAs were identified and confirmed by APCI-MS. The main components of three Compositae plants were also compared. Although there exist some similarities in the flavonoidic content of the leaf and flower of C. morifolium, significant variations in their varities and concentrations were observed. Artemisia annua processes substantial amount of alkylated derivatives of flavones and Chrysanthemum coronarium contains only CQAs. These findings suggest that although all the plants studied are from the same Compositae family, their flavonoids and phenolic compositions are markedly different. The proposed method is useful for further chromatographic fingerprinting of plant flavonoids.
Keywords: LC/DAD-APCI/MS; Flavonoids; Caffeoylquinic acids; Compositae plant; Chromatographic fingerprinting;

A competitive immunoassay for detecting clenbuterol in urine was established by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). The clenbuterol was conjugated with bovine serum albumin (BSA), and then the derivative was labeled with fluorescein isothiocyanate (FITC) and competes for antibody with free clenbuterol in the sample. Under the optimal conditions, Free and bound FITC labeled clenbuterol was separated within 8 min with the relative standard deviation (R.S.D.) 0.72% for migration time and 2.8% for peak area. The detection limit reached 0.7 ng/ml.
Keywords: Clenbuterol; Capillary electrophoresis; Immunoassay;

Estimation of boswellic acids from market formulations of Boswellia serrata extract and 11-keto β-boswellic acid in human plasma by high-performance thin-layer chromatography by Shailesh A. Shah; Ishwarsinh S. Rathod; Bhanubhai N. Suhagia; Dharmesh A. Patel; Vijay K. Parmar; Bharat K. Shah; Vikas M. Vaishnavi (232-238).
A rapid and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the quantitative estimation of boswellic acids in formulation containing Boswellia serrata extract (BSE) and 11-keto β-boswellic acid in human plasma. Simple extraction method was used for isolation of boswellic acid from formulation sample and acidified plasma sample. The isolated samples were chromatographed on silica gel 60F254-TLC plates, developed using ternary-solvent system (hexane–chloroform–methanol, 5:5:0.5, v/v) and scanned at 260 nm. The linearity range for 11-KBA spiked in 1 ml of plasma was 29.15–145.75 ng with average recovery of 91.66%. The limit of detection and limit of quantification for 11-KBA in human plasma were found to be 8.75 ng/ml and 29.15 ng/ml. The developed method was successfully applied for the assay of market formulations containing BSE and to determine plasma level of 11-keto β-boswellic acid in a clinical pilot study.
Keywords: Boswellia serrata extract; 11-Keto boswellic acid; Market formulation; Human plasma; HPTLC method;

An automated high-throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed for quantitative determination of donepezil in human plasma. 150 μL of plasma samples were placed in 2.2 mL 96-deepwell plates and both donepezil and loratadine (IS) were extracted from human plasma by liquid–liquid extraction (LLE), using hexane as the organic solvent. Robotic liquid handling workstations were employed for all liquid transfer and solution preparation steps and resulted in a short sample preparation time. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted in a small volume of reconstitution solution. The method developed, includes a sample analysis performed by reversed phase LC–MS/MS, with positive ion electrospray ionization, using multiple reaction monitoring (MRM). The chromatographic run time was set for 2.0 min with a flow rate of 0.7 mL/min in a C18 analytical column. The method was significantly sensitive, specific, accurate and precise for the determination of donepezil in human plasma and had the shortest run time. The curve was proven to be linear for the concentration range of 0.1–100 ng/mL. After validation, the method was applied to the rapid and reliable quantitative determination of donepezil in a bioequivalence study after per os administration of a 5 mg donepezil tablet.
Keywords: Donepezil; Bioequivalence; 96-Well format; LC–MS/MS;

Liquid chromatographic determination of irbesartan in human plasma by Ashok K. Shakya; Yusuf M. Al-Hiari; Omran M.O. Alhamami (245-250).
A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm × 4.6 mm i.d., particle size 5 μm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was ∼13 min. The validated quantitation ranges of this method were 15–4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3–77.1% with coefficients of variation of 3.7–6.3%. The between- and within-batch precision were 0.4–2.2% and 0.9–6.2%, respectively. The between- and within-batch relative errors (bias) were (−5.5) to 0.9% and (−0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at −70 °C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.
Keywords: Irbesartan; Fluorescence detection; Liquid–liquid extraction;

Quantification of free and total sialic acid excretion by LC–MS/MS by Maria van der Ham; Berthil H.C.M.T. Prinsen; Jan G.M. Huijmans; Nicolaas G.G.M. Abeling; Bert Dorland; Ruud Berger; Tom J. de Koning; Monique G.M. de Sain-van der Velden (251-257).
The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC–MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated.FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared. 13C3-sialic acid was used as internal standard. LC–MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA.Limit of detection for FSA and TSA was 0.3 and 1.7 μmol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 μmol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n  = 10) for FSA and 6.5% and 3.6% (n  = 10) for TSA. Linearity was tested till 7800 μmol/L (r 2  = 0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values.A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.
Keywords: Sialic acid; Salla disease; Sialuria; Sialidosis; Galactosialidosis; Hemolytic uremic syndrome;

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain by Richard Tykva; Jan Hlaváček; Věra Vlasáková; Bohuslav Černý; Lenka Borovičková; Blanka Bennettová; Josef Holík; Jiřina Slaninová (258-263).
Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80–150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.
Keywords: Peptide synthesis; Radiolabeling; Radio-HPLC; Course of degradation; Tyrosine metabolism;

A method was developed for quantification of oxycodone, noroxycodone, and oxymorphone in small volumes (50 μl) of rat plasma by high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry using turbo ion-spray. Deuterated (d3) opioid analogues acted as internal standards. Sample preparation involved protein precipitation with acetonitrile, centrifugal evaporation, and reconstitution in mobile phase; analyte separation was performed on a C18 (5 μm, 2.1 mm × 50 mm) column using a linear gradient program. Lower limits of quantitation (ng/ml) and their between-day accuracy and precision were—oxycodone, 0.9 (−0.2 and 7.8%); noroxycodone, 1.0 (0.6 and 6.2%); oxymorphone 1.0 (−1.8 and 9.5%).
Keywords: Oxycodone; Noroxycodone; Oxymorphone; Rat; High-performance liquid chromatography (HPLC); Electrospray ionization (ESI); Tandem mass spectrometry (MS-MS);

A simple and rapid HPLC/UV method for the simultaneous quantification of theophylline and etofylline in human plasma by Ramakrishna V.S. Nirogi; Vishwottam N. Kandikere; Manoj Shukla; Koteshwara Mudigonda; Devender R. Ajjala (271-276).
A simple, sensitive and selective high performance liquid chromatography (HPLC) method with ultraviolet detection (272 nm) was developed and validated for the simultaneous quantification of theophylline and etofylline in human plasma. Following rapid sample preparation, the analytes and internal standard (hydrochlorothiazide) were separated using an isocratic mobile phase on a reverse phase C18 column. The lower limit of quantification was 100 ng/mL for both theophylline and etofylline with a relative standard deviation of less than 6%. A linear dynamic range of 100–10,000 ng/mL for both theophylline and etofylline was established. This HPLC method was validated with between-batch precision of 2.2–6.0 and 1.4–3.7% for theophylline and etofylline, respectively. The between-batch accuracy was 94.3–98.0 and 95.4–98.2%, respectively. Stability of theophylline and etofylline in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is simple and rugged enough to be used in pharmacokinetic studies.
Keywords: Theophylline; Etofylline; Simultaneous quantification in human plasma; HPLC;

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 °C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and −20 °C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2–3 h after the end of exposure.
Keywords: Headspace technique; Trihalomethanes (THMs); Swimming pools; Urine sample; Exposure;

Determination of long-chain fatty acids in bryophyte plants extracts by HPLC with fluorescence detection and identification with MS by Jinmao You; Xianen Zhao; Yourui Suo; Yulin Li; Honglun Wang; Guichen Chen (283-291).
A sensitive method for the determination of long-chain fatty acids (LCFAs) (>C20) using 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) as tagging reagent with fluorescence detection and identification with post-column APCI/MS has been developed. The LCFAs in bryophyte plant samples were obtained based on distillation extraction with 1:1 (v/v) chloroform/methanol as extracting solvent. TSPP could easily and quickly label LCFAs at 90 °C in the presence of K2CO3 catalyst in DMF. Eleven free LCFAs from the extracts of bryophyte plants were sensitively determined. Maximal labeling yields close to 100% were observed with a five-fold excess of molar reagent. Separation of the derivatized fatty acids exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Eclipse XDB-C8 column. Calculated detection limits from 1.0 pmol injection, at a signal-to-noise ratio of 3, were 26.19–76.67 fmol. Excellent linear responses were observed with coefficients of >0.9996. Good compositional data were obtained from the analysis of the extracted LCFAs containing as little as 0.2 g of bryophyte plant samples. Therefore, the facile TSPP derivatization coupled with HPLC/APCI/MS analysis allowed the development of a highly sensitive method for the quantitation of trace levels of LCFAs from biological and natural environmental samples.
Keywords: 1-[2-(p-Toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP); Derivatization; HPLC; Fatty acids; Mass spectrometry;

Direct-injection LC–LC hybrid tandem MS methods have been developed for undertaking broad-based screening for acidic drugs in protein-precipitated plasma and neutral doping agents in equine urine. In both analyses, analytes present in the matrix were trapped using a HLB® extraction column before being refocused and separated on a Chromolith® RP-18e monolithic analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Each method has been optimised by the adoption of a mobile phase and gradient that was tailored to enhance ionisation in the MS source while maintaining good chromatographic behaviour for the majority of the target drugs. The analytical column eluent was fed into the heated nebulizer (HN) part of the Duospray® interface attached to a 4000 QTRAP® mass spectrometer. Information dependent acquisition (IDA) with dynamic background subtraction (DBS) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan signal exceeded the defined criteria. Ninety-one percent of acidic drugs in protein-precipitated plasma and 80% of the neutral compounds in equine urine were detected when spiked at 10 ng/ml.
Keywords: Direct injection; Hybrid MS/MS; Equine drug testing; Differential LC/LC gradient;

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection by Michael Jason Bishop; Brian S Crow; Kasey D Kovalcik; Joe George; James A Bralley (303-310).
A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r 2  ≥ 0.995), accuracy (85–115%), precision (C.V. < 12%), sample preparation stability (≤5%, 72 h), and established patient ranges. The method was found to be both efficient and accurate for the analysis of urinary zwitterionic organic acids.
Keywords: Weak Anion Exchange; Formiminoglutamic acid; Pyroglutamic acid; 5-hydroxyindoleacetic acid; 2-methylhippuric acid; LC/MS/MS; Organic acids;

Determination of memantine in human plasma by liquid chromatography–electrospray tandem mass spectrometry: Application to a bioequivalence study by A.A. Almeida; D.R. Campos; G. Bernasconi; S. Calafatti; F.A.P. Barros; M.N. Eberlin; E.C. Meurer; E.G. Paris; J. Pedrazzoli (311-316).
A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of memantine (I) in human plasma is presented. Sample preparation consisted of the addition of amantadine (II) as internal standard (IS), liquid–liquid extraction in basic conditions using a mixture of diethyl ether–chloroform (7:3, v/v) as extracting solvent, followed by centrifugation, solvent evaporation and sample reconstitution in methanol. Both I and II (internal standard) were analyzed using a C18 column and a mobile phase composed of methanol–water–formic acid (80:20:0.1, v/v/v). Eluted compounds were monitored using positive mode electrospray (ES) tandem mass spectrometry. The analyses were carried out by selected reaction monitoring (SRM) using the parent to daughter combinations of m/z 180 > 163 (memantine) and m/z 152 > 135 (amantadine). The peak areas from the analyte and IS were used for quantification of I. The achieved limit of quantification (LOQ) was 0.1 ng/mL; the assay exhibited a linear dynamic range of 0.1–50.0 ng/mL with a determination coefficient (r 2) of at least 0.98. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of samples taken up to 320 h after oral administration of 20 mg (two 10 mg capsules) of I in healthy volunteers demonstrated the applicability to bioequivalence studies.
Keywords: Mass spectrometry; Bioequivalence; Memantine;

A LC-MS/MS method was developed for quantitative determination of esomeprazole, and its two main metabolites 5-hydroxyesomeprazole and omeprazole sulphone in 25 μL human, rat or dog plasma. The analytes and their internal standards were extracted from plasma into methyl tert-butyl ether - dichloromethane (3:2, v/v). After evaporation and reconstitution of the organic extract the analytes were separated on a reversed-phase LC column and measured by atmospheric-pressure positive ionisation MS. The linearity range was 20–20,000 nmol/L for esomeprazole and omeprazole sulphone, and 20–4000 nmol/L for 5-hydroxyesomeprazole. The extraction recoveries ranged between 80 and 105%. The intra- and inter-day imprecision were less than 9.5% with accuracy between 97.7% and 100.1% for all analytes.
Keywords: LC-MS/MS; Esomeprazole; Metabolites;

Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid by A.J. Devall; R. Blake; N. Langman; C.G.S. Smith; D.A. Richards; K.J. Whitehead (323-328).
The development of a HPLC method using a monolithic C18 column is described using fluorescence detection for the assay of 21 amino acids and related substances with derivatisation using ortho-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA). The method employs a tertiary gradient and has a run time of 24 min. Linearity (r 2) for each amino acid was found to be greater than 0.99 up to a 10 μM concentration; reproducibility across all analyses (relative standard deviation (R.S.D.)) was between 0.97 and 6.7% and limit of detection (LOD) between 30 and 300 fmol on column. This method has been applied to the analysis of amino acids in both spinal microdialysis and cerebral spinal fluid samples.
Keywords: Reversed-phase liquid chromatography; Amino acid; Monolithic; Ortho-phthaldialdehyde;

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 μg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.
Keywords: Valganciclovir; Ganciclovir; Aciclovir; LC/MS/MS; Protein precipitation;

The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2′-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable.
Keywords: Nucleotide; LC/MS/MS; Ion pairing; Adefovir; Hepatitis B virus; Metabolism;

A fast, sensitive and specific LC–MS/MS bioanalytical method for the determination of unchanged clopidogrel in human plasma has been developed and validated over the range of 10–12,000 pg mL−1 (r 2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 μL of the sample extract was injected onto the LC–MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 °C) by gradient elution at a flow rate of 0.9 mL min−1 over a 3 min run time. Retention times of 1.61 and 1.59 min were observed for clopidogrel and 2H3-clopidogrel (I.S.), respectively. Detection was achieved using a Sciex API 4000, triple quadrupole mass spectrometer, in positive TurboIonspray™ (electrospray) ionisation mode. Ion transitions were monitored using MRM (multiple reaction monitoring) for clopidogrel (m/z 322–212) and for 2H3-clopidogrel (m/z 327–217). This validated method was used to support a pharmacokinetic study in healthy volunteers.
Keywords: LC–MS/MS; Validation; Clopidogrel; Human plasma; Bioanalytical;

The root of Scutellaria baicalensis, called Huangqin in Chinese, is one of the most commonly used traditional Chinese medicines for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. The major chemical constituents of Huangqin are flavonoids. In the present paper, HPLC-DAD-ESI-MS n was used to analyze flavonoids in the roots of S. baicalensis. A total of 26 flavonoids were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides, and 9 free aglycones. Two C-glycosides, apigenin-6-C-glucyl-8-C-arabinoside and chrysin-6,8-di-C-glucoside, together with some O-glycosides, are reported from S. baicalensis for the first time. This method is simple, reliable and sensitive, and could be used for the quality control of Huangqin and its related preparations.
Keywords: Huangqin; Flavonoids; HPLC/DAD/ESI-MS n ; Chinese medicine;

Determination of protodioscin in rat plasma by liquid chromatography–tandem mass spectrometry by Tiejie Wang; Zhongbo Liu; Jun Li; Min Zhong; Junpeng Li; Xiaohui Chen; Kaishun Bi (363-368).
Protodioscin (3-O-[α-l-rhamnopyranosyl-(1 → 2)-{α-l-rhamnopyranosyl-(1 → 4)}-β-d-glucopyranosyl]-26-O-[β-d-glucopyranosyl]-(25 R)-furost-5-ene-3 β,26-diol) is a naturally occurring saponin present in many oriental vegetables and traditional medicinal plants, which has been associated with potent bioactivity. However, there is no specific and sensitive assay for quantitative determination of protodioscin in biological samples. We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples were prepared after plasma protein precipitation, and a aliquot of the supernatant was injected directly onto an analytical column with a mobile phase consisted of acetonitrile–water–formic acid (80:20:0.1, v/v/v). Analytes were detected with a LC-ESI-MS/MS system in positive selected multiple reaction-monitoring mode. The lower limit of quantification (LLOQ) was 20.0 ng/mL and a linear range of 20–125,000 ng/mL. The intra- and inter-day relative standard deviation (R.S.D.) across three validation runs over the entire concentration range was <8.0%. Accuracy determined at three concentrations (50, 5000 and 50,000 ng/mL for protodioscin) ranged from 0.2 to 1.8% as terms of relative error (R.E.). Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma.
Keywords: Liquid chromatography; Mass spectrometry; Protodioscin; Pharmacokinetics;

An efficient, isocratic high performance liquid chromatography (HPLC) method for determining human immunodeficiency virus (HIV) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in plasma is advantageous for laboratories participating in clinical trials and therapeutic drug monitoring (TDM) programs, or conducting small animal research. The combination of isocratic reversed phase chromatography using an S-3, 3.0 mm × 150 mm column along with low plasma volume (200 μl), rapid liquid–liquid extraction, and detection at a single wavelength (212 nm) over a short run time makes this method valuable. Within and between assay variability ranges from 0.8 to 3.5% and 1.2–6.2%, respectively. Accuracy ranges from 91.0 to 112.8% for four quality controls (50, 100, 1000, and 10,000 ng/ml) for all drugs measured (efavirenz, nevirapine, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir).
Keywords: HIV; Non-nucleoside reverse transcriptase inhibitors; Protease inhibitors; HPLC-UV; Isocratic; Liquid–liquid extraction; Therapeutic drug monitoring;

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method by Antonio D’Avolio; Mauro Sciandra; Marco Siccardi; Lorena Baietto; Daniel Gonzalez de Requena; Stefano Bonora; Giovanni Di Perri (374-378).
A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 μl of plasma before a liquid–liquid extraction by 600 μl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from −1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 μg/ml and 0.035 μg/ml, respectively. Mean recovery was 87.1% ± 2.4%. Calibration curve was linear up to 180 μg/ml. Concentration range when optimized (0.703–180 μg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.
Keywords: Tipranavir; UV detector; Liquid/liquid extraction; TDM;

An accurate and precise method was developed using HPLC-MS/MS to quantify erlotinib (OSI-774) and its O-desmethyl metabolite, OSI-420, in plasma. The advantages of this method include the use of a small sample volume, liquid–liquid extraction with high extraction efficiency and short chromatographic run times. The analytes were extracted from 100 μL plasma volume using hexane:ethyl acetate after midazolam was added to the sample for internal standardization. The compounds were separated on a Phenomenex C-18 Luna analytical column with acetonitrile:5 mM ammonium acetate as the mobile phase. All compounds were monitored by tandem mass spectrometry with electrospray positive ionization. The intra-day accuracy and precision (% coefficient of variation, % CV) estimates for erlotinib at 10 ng/mL were 90% and 9%, respectively. The intra-day accuracy and precision estimates for OSI-420 at 5 ng/mL were 80% and 4%, respectively. This method was used to quantify erlotinib and OSI-420 in plasma of patients (n  = 21) administered 150 mg erlotinib per day for non-small cell lung cancer.
Keywords: Erlotinib; OSI-774; HPLC-MS/MS;

Traces of phosgene in chloroform: Consequences for extraction of anthracyclines by Kristof E. Maudens; Sarah M.R. Wille; Willy E. Lambert (384-390).
Chloroform is commonly used to extract anthracyclines from various biological matrices. However, their determination can be seriously compromised by phosgene traces present as a result of failing stabilization of chloroform. Out of the three varieties in which chloroform exists (not stabilized, stabilized with an alcohol and stabilized with a hydrocarbon) only the ethanol stabilized type minimizes chances on creating artifacts. Chromatographic separation after extraction of four anthracyclines (doxorubicin, epirubicin, daunorubicin and idarubicin) and two metabolites (13-S-dihydrodoxorubicin and 13-S-dihydroepirubicin) with chloroform under various conditions indicate that the appropriate choice of stabilizer in this extraction solvent is highly relevant.
Keywords: Chloroform; Stability; Phosgene; Ethanol; Amylene; 2-Methyl-2-butene; Anthracyclines; Liquid–liquid extraction;

We established a method for automated quantitative analysis of (es-)citalopram and desmethyl(es-)citalopram in serum using column-switching high performance liquid chromatography (HPLC). For sample clean-up serum was injected onto a LiChrospher CN 20 μm precolumn using 8% acetonitrile in deionized water. Drugs were eluted by back-flush flow onto the analytical column (LiChrospher CN 5 μm) at a flow rate of 1.5 ml/min with phosphate buffer 8 mmol/l pH 6.4/acetonitrile (50/50, v/v). Haloperidol was used as internal standard. Analytes were detected by ultraviolet spectrophotometry at 210 nm. Detection limit of (es-)citalopram was 6 ng/ml. The method was found to be suitable for therapeutic drug monitoring of patients treated with citalopram or escitalopram.
Keywords: Citalopram; Escitalopram; Quantitative analysis; HPLC column-switching; TDM;