Journal of Pharmaceutical and Biomedical Analysis (v.61, #C)

► GDC-0941 is a small molecule class I phosphatidylinositide 3-kinase inhibitor. ► A SPE-LC–MS/MS analytical method was validated for GDC-0941. ► Validation met criteria of the “Guidance for Industry” set by the FDA. ► Pharmacokinetic results of a phase I clinical trial are presented.A solid phase extraction (SPE) liquid chromatographic–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC-0941 concentrations in human plasma has been developed and validated to support clinical development. An Oasis MCX 10 mg 96-well SPE plate was used to extract plasma samples (50 μL) and the resulting extracts were analyzed using reverse-phase chromatography and mass spectrometer coupled with a turbo-ionspray interface. The method was validated over the calibration curve range 0.500–500 ng/mL with linear regression and 1/x 2 weighting. Within-run relative standard deviation (%RSD) ranged from 1.5 to 11.5%, while the between-run %RSD varied from 0.0 to 4.4%. The accuracy ranged from 96.0% to 110.0% of nominal for within-run and 98.0% to 108.0% of nominal for between-run at all concentrations including the LLOQ quality control at 0.500 ng/mL. Extraction recovery of GDC-0941 was between 79.0% and 86.2%. Stability of GDC-0941 was established in human plasma for 602 days at −70 °C and 598 days at −20 °C, respectively, and established in reconstituted sample extracts for 167 h when stored at room temperature. Internal standard normalized matrix factor was 1.1, demonstrating that the use of the stable-labeled internal standard GDC-0941-d8 effectively compensated observed matrix effect and resulting in no adverse impact on the quality of the data produced. This assay was used for the determination of GDC-0941 human plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.
Keywords: GDC-0941; Class I phosphatidylinositide 3-kinase (PI3K); 96-Well SPE; Human plasma; LC–MS/MS;

Oral and topical pharmacokinetic studies of a novel TRPV1 antagonist, PAC-14028 in rats and minipigs using liquid chromatography/tandem mass spectrometric method by Yang-Hui Park; Kyung-Mi Joo; Byoung-Young Woo; Eui Dong Son; Sang Yo Byun; Hong-Ju Shin; Ki-Wha Lee; Young-Ho Park; Kyung-Min Lim (8-14).
PAC-14028 ((E)-N-((R)-1-(3,5-difluoro-4-methanesulfonylamino-phenyl)-ethyl)-3-(2-propyl-6-trifluoromethyl-pyridine-3-yl)-acrylamide) is a novel and potent transient receptor potential vanilloid type I (TRPV1) antagonist. We developed and validated a rapid, sensitive and selective liquid chromatography/tandem mass spectrometric method for determination of PAC-14028 in rat and minipig plasma. After protein precipitation PAC-14028 and internal standard (methylated analog, PAC-14026) were separated on a Symmetry C18 column (4.6 mm × 75 mm, 3.5 μm) with an isocratic mobile phase, acetonitrile: water (8:2, v/v) containing 0.2% formic acid and monitored by electrospray positive ionization with multiple reaction monitoring mode (PAC-14028, 492 → 156; IS, 506 → 156, m/z). The calibration curve was linear over the range of 1.0–500 ng/ml (r 2  > 0.999) and lower limit of quantitation (LLOQ) was 1 ng/ml. The precision and accuracy were within ±15% and the stability was acceptable during bench-top, auto-sampler, 3 freeze–thaw cycles and 4-week storage in a freezer at −80 °C. This method was successfully applied to the intravenous, oral and topical pharmacokinetic studies of PAC-14028 in rats and minipigs, which showed comparable pharmacokinetic parameters (T1/2, 2.1 h and 3.8 h; F%, 52.7% and 64.2% for rats and minipigs, respectively). Percutaneous absorption of PAC-14028 was negligible after topical application (F% 0.2–1.7%).
Keywords: Transient receptor potential vanilloid type I; PAC-14028; Pharmacokinetic; LC/MS/MS; Topical;

► An LC-MS assay for hydromorphone (QMFT) in human plasma was established with an LLOQ of 0.01 ng/mL. ► The method was successfully applied to the PK study of QMFT in postoperative patients. ► The pharmacokinetics of QMFT in postoperative patients was reported for the first time. ► The chromatographic peak shape deterioration was solved by increasing the column temperature. ► The MS response was improved by reducing the flow rate.A sensitive reverse phase high performance liquid chromatography–electrospray ionization-mass spectrometry (RP-HPLC–ESI-MS) method has been developed and validated for the determination of hydromorphone in human plasma using naloxone as the internal standard (IS). After alkalization with saturated sodium bicarbonate, the plasma samples were extracted with ethyl acetate. Chromatographic separation was performed on a C18 column with the column temperature of 50 °C and a mobile phase of 5 mM ammonium acetate buffer containing 1% formic acid–methanol (88:12, v/v). Hydromorphone and the IS were detected by selected ion monitoring using the protonated molecules at m/z 286.2 for hydromorphone and m/z 328.2 for the IS. Calibration curve was linear over the range of 0.01–50 ng/mL. The lower limit of quantification was 0.01 ng/mL. The method was successfully applied to the pharmacokinetic study in postoperative patients after intravenous infusion of 1.5 mg hydromorphone hydrochloride. The obtained main pharmacokinetic parameters of hydromorphone in postoperative patients were as follows: the maximum hydromorphone plasma concentration (C max) was (24.15 ± 12.51) ng/mL, the time to the C max was (10.0 ± 0.0) min, and the elimination half-life was (2.7 ± 0.8) h.
Keywords: Hydromorphone; Pharmacokinetics; RP-HPLC–ESI-MS; Postoperative patient;

In vivo microdialysis with LC–MS for analysis of spinosin and its interaction with cyclosporin A in rat brain, blood and bile by Rong-Hua Ma; Jie Yang; Lian-Wen Qi; Gui-Zhong Xin; Chong-Zhi Wang; Chun-Su Yuan; Xiao-Dong Wen; Ping Li (22-29).
► Analysis of hepatobiliary excretion and brain metabolism is important to understanding drug metabolism. ► Technical difficulties limited spinosin analysis in rat blood, bile and brain. ► A microdialysis technique with HPLC–MS method was developed to investigate the pharmacokinetics of unbound spinosin in the brain, blood and bile dialysate of rats, this method can be conveniently and sensitively applied to monitor the pharmacokinetic behavior of drugs.Spinosin, a major bioactive herbal ingredient isolated from Semen Ziziphi Spinosae, plays an important role in sedation and hypnosis. However, the pharmacokinetic behavior of spinosin in special sites has not been reported. Microdialysis (MD) technique, as a continuous, realtime monitoring sampling technique, is very suitable for the evaluation of the disposition of diverse drugs. To obtain more useful information on spinosin, an in vivo microdialysis sampling technique with High Performance Liquid Chromatography–mass spectrograph (HPLC–MS) method was developed to investigate the pharmacokinetics of spinosin and its interaction with cyclosporin A (CsA) in the brain, blood and bile of rats. The method was validated in terms of selectivity, linearity and sensitivity, and showed advantages in monitoring the pharmacokinetic behavior of drugs. The results revealed that CsA has obvious effects on the pharmacokinetic process of spinosin. When co-administered, the area under the curve (AUC) of spinosin in blood, bile and brain increased from 205.70 to 673.51 mg min/L, 7.77 × 104 to 1.25 × 105  mg min/L, and 2.09 to 5.58 mg min/L, respectively. The t 1/2 values of spinosin in blood, bile and brain also changed from 48.07 to 95.04 min, from 97.20 to 152.21 and from 42.18 to 73.83 min, respectively. These results demonstrated that the CsA decreased the efflux of spinosin through the inhibition of P-glycoprotein (P-gp) efflux transporter and it might be used as a group of P-gp substrate. Other transporters or pathways may also be involved in the metabolism of spinosin.
Keywords: Spinosin; In vivo microdialysis; LC–/MS; Cyclosporin A; Drug–drug interactions;

► Severe interferences were observed when DBS sampling was used in LC/MS/MS analysis. ► It was determined that SDS coated on DMPK-A cards formed ion pairs with compounds containing basic amine groups. ► Non-acidic mobile phases and DBS cards with no SDS coating alleviated the interferences. ► DBS users should always be aware of possible analyte interactions with DBS card constituents.Unique and remarkable interferences were observed when dried blood spot (DBS) sampling was used in conjunction with liquid chromatography/mass spectrometry (LC/MS) assays. In particular, chromatographic retention time shifting and chromatographic peak shape distortion were observed, along with a severe suppression of MS signal intensity. The type of DBS cards, and chromatographic conditions were investigated using the same set of test compounds to gain insight into these interferences. It was determined that a constituent of the DBS cards, primarily sodium dodecyl sulfate (SDS), was responsible for the interferences by means of an ion-pairing mechanism. SDS formed ion pairs with compounds containing basic amine groups, which resulted in increased retention on a C18 stationary phase, peak shape distortion and ion suppression. These interferences were greatly alleviated and/or completely overcome with non-acidic mobile phases and/or DBS cards with no SDS coating. To the best of the authors’ knowledge, this is the first in-depth report of interferences induced by DBS cards.
Keywords: Liquid chromatography/mass spectrometry; Dried blood spotting; Interference; Ion-pairing; FTA® DMPK-A card;

A rapid and sensitive HPLC–APCI-MS/MS method determination of fluticasone in human plasma: Application for a bioequivalency study in nasal spray formulations by Ricardo Martins Duarte Byrro; Isabela Costa César; Fabiana Fernandes de Santana e Silva Cardoso; Iram Moreira Mundim; Leonardo de Souza Teixeira; Ricardo Rodrigues Bonfim; Sandro Antônio Gomes; Gerson Antônio Pianetti (38-43).
► Bioequivalence study of fluticasone administered by nasal spray formulations. ► A sensitive LC–API-MS/MS method for the determination of fluticasone in plasma. ► Liquid–liquid extraction sample preparation. ► Sensitive method with lower limit of quantification set at 2 pg/mL.A sensitive method for the determination of fluticasone in plasma was developed using high performance liquid chromatography with tandem mass spectrometric detection, whereas beclomethasone was used as internal standard. The analytes were extracted with a simple liquid–liquid extraction from the plasma samples and separated on an ACE C18 50 × 4.6 mm i.d.; 5 μm particle size column with a mobile phase consisting of acetonitrile – 0.01% formic acid (48:52, v/v) at a flow rate of 1 ml/min. Detection was achieved by an Applied Biosystems API 5000 mass spectrometer (LC–MS/MS) set at unit resolution in the multiple reaction monitoring mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for fluticasone propionate was 85%, with a lower limit of quantification set at 2 pg/mL. The validated analytical method was applied to a bioequivalence study of fluticasone propionate administered by nasal spray formulations in human volunteers.
Keywords: Fluticasone; Nasal spray; HPLC–APCI-MS/MS; Plasma; Bioequivalence;

A simple, inexpensive and efficient sample preparation technique, three-phase hollow fiber liquid phase microextraction (HF-LPME), followed by high-performance liquid chromatography–ultra violet detection (HPLC–UV) was used for the analysis of warfarin in human plasma. Warfarin was extracted from 11.0 ml of aqueous solution with pH = 2.3 (donor phase) into 1-octanol immobilized in the wall pores of a porous hollow fiber and then extracted into the acceptor phase with pH = 11.0 located in the lumen of the hollow fiber. After the extraction, the acceptor phase was directly injected into the HPLC system for quantification. Different factors affecting the HF-LPME including nature of organic extraction solvent, pH of donor and acceptor phases, stirring rate, extraction time and salt addition to the sample solution (donor phase) were investigated and optimized. Under the optimized conditions (1-octanol as organic solvent, pHdonor  = 2.3, pHacceptor  = 11.0, stirring speed of 1000 rpm, extraction time of 30 min, without addition of salt), limit of detection (LOD) of 5 ng ml−1 and enrichment factor of 225 were obtained. The calibration curve was linear within the range of 15–550 ng ml−1 with the square of correlation coefficient of 0.9984. The values of intra-day relative standard deviation (RSD) and inter-day RSD were 4.2% and 11.1%, respectively. The method was applied successfully for the analysis of warfarin in plasma sample from a patient under treatment with this drug and excellent sample clean-up was observed.
Keywords: Hollow fiber; Liquid phase microextraction; Warfarin; HPLC; Plasma;

A sensitive and specific high performance liquid chromatography–ultraviolet detection (HPLC–UV) method for quantitative determination of exo- and endo-iohexol in cat and dog serum/plasma is presented.Sample preparation consisted of a protein precipitation step performed by adding 15 μL of trifluoroacetic acid to 100 μL of serum/plasma. Following vortexing and centrifugation, an aliquot of the supernatant was injected onto a polymeric PLRP-S column (250 mm × 4.6 mm i.d., dp: 8 μm, 100 Å), maintained at 30 °C. The mobile phase consisted of water (A) and methanol (B) and a gradient elution (flow-rate: 1.0 mL min−1, total run-time: 21 min). The UV detector was set at a wavelength of 254 nm.Matrix-matched calibration graphs were prepared for both exo- (0.44–657 μg mL−1) and endo-iohexol (0.62–93.0 μg mL−1). Correlation and goodness-of-fit coefficients were between 0.9985–0.9999 and 4.44–9.87%, respectively. Limits of quantification and detection were 0.44 and 0.15 μg mL−1 for exo-iohexol and 0.62 and 0.20 μg mL−1 for endo-iohexol, respectively. Results for within-day and between-day precision and accuracy fell within the ranges specified.The reported method is simple and cost-effective. It has been successfully used for the analysis of exo- and endo-iohexol in serum/plasma samples of cats and dogs as part of pharmacokinetic studies with iohexol in order to determine plasma clearance of exo- and endo-iohexol. This indicates the usefulness of the developed method for application in the field of veterinary clinical practice and research.
Keywords: Iohexol; Cat; Dog; HPLC–UV; Plasma;

► Complete baseline separation of tacrine and its three metabolites was achieved. ► Orally administered tacrine was quickly absorbed and extensive metabolized in rat. ► Tacrine and its monohydroxylated metabolites were evenly distributed in rat brain. ► Tacrine and 4-hydroxytacrine exhibited much higher brain-to-plasma ratios.The current study aims to investigate the pharmacokinetics and brain disposition of tacrine and its three major bioactive monohydroxylated metabolites (1-hydroxytacrine, 2-hydroxytacrine and 4-hydroxytacrine). An assay for simultaneous quantification of tacrine and three above metabolites in rat plasma and brain tissue was developed. Four analytes together with internal standard were extracted from rat plasma or brain tissue homogenate by liquid–liquid extraction using ethyl acetate. Baseline separation of the four studied compounds was achieved by a Thermo Hypersil BDS C18 column with gradient elution using acetonitrile and ammonium formate-triethylamine (pH 4.0) under fluorescence detection. Extraction recoveries of all analytes ranged from 82.1% to 93.2% in both rat plasma and brain tissue. The intra- and inter-day precision and accuracy of each analyte at lower limit of quantification (LLOQ) and three quality control (QC) concentrations (low, middle and high) was within 12% RSD and within 11% bias in both biological matrices. The LLOQ for tacrine, 1-hydroxytacrine, 2-hydroxytacrine and 4-hydroxytacrine were found to be 2.5, 6.7, 2.1 and 2.1 ng/ml in plasma, and 12.3, 33.5, 10.6 and 10.5 ng/g in brain tissue, respectively. All four analytes were stable during analysis. The developed method provides a simple, sensitive and reproducible procedure for pharmacokinetics and brain disposition study of tacrine and its three major metabolites in rats after oral administration of tacrine. Pharmacokinetic study demonstrated that tacrine could be quickly absorbed and extensively metabolized to its monohydroxylated metabolites in vivo. In addition, rat brain disposition study showed that tacrine and its monohydroxylated metabolites were evenly distributed in different brain regions except for a slight lower concentration of tacrine in olfactory region. Moreover, tacrine and 4-hydroxytacrine exhibited a much higher brain-to-plasma ratio than that of 1-hydroxytacrine and 2-hydroxytacrine.
Keywords: Tacrine; Hydroxytacrine; Pharmacokinetics; Brain disposition; Fluorescence;

Quantitative analysis of mephedrone using liquid chromatography tandem mass spectroscopy: Application to human hair by Syeda A.B. Shah; Nawed I.K. Deshmukh; James Barker; Andrea Petróczi; Paul Cross; Roland Archer; Declan P. Naughton (64-69).
► The study describes a rapid and replicable method for the quantitative analysis of mephedrone and its two metabolites in human hair. ► The method is highly selective and sensitive with an LOQ of 5 pg/mg for mephedrone and 10 pg/mg for its two metabolites. ► Five out of 154 hair samples were confirmed to be positive for mephedrone.Recent abuse of designer drugs such as mephedrone has presented a requirement for sensitive, reliable and reproducible methods for the detection of these controlled drugs in different matrices. This study focuses on a fully developed validated method for the quantitative analysis of mephedrone and its two metabolites 4-methylephedrine and 4-methylnorephedrine in human hair. The calibration curve was found to be linear in the range 5–100 pg/mg for mephedrone and 10–150 pg/mg for 4-methylephedrine and 4-methylnorephedrine. The method was successfully validated for the intraday precision, interday precision, limit of detection, accuracy and extraction recovery. Five out of 154 hair samples were confirmed to be positive for mephedrone. Due to the structural similarities to other methcathinones and amphetamines, one can propose the metabolism for mephedrone based on a similar pathway that has been previously used for these psychoactive drugs. The outlined method can be valuable for the future detection of mephedrone and its two metabolites in hair.
Keywords: Mephedrone; Metabolites; 4-Methylephedrine; 4-Methylnorephedrine: LC–MS/MS;

► Alamifovir disoproxil fumarate (ADF) is developed as a promising anti-HBV candidate. ► The anti-HBV activity of ADF is 100 times higher than that of alamifovir. ► The oral bioavailability of ADF is about 3 times higher than that of alamifovir.Alamifovir disoproxil fumarate (ADF) is a novel ester prodrug of alamifovir, which is currently as a promising antiviral candidate under investigation. This paper is aimed to develop rapid, sensitive and specific LC–MS/MS methods for the quantification of ADF and its active metabolite 602076 in rat plasma. According to the significantly different chemical properties of the compounds, two sets of liquid chromatography and ionization modes were used for determining the concentration of ADF and 602076 in rat plasma, separately. Following liquid–liquid extraction with n-hexane:dichloromethane:isopropanol (100:50:5, v/v/v), the ADF and internal standard (gliclazide) were separated on a Phenomenex Gemini C18 column (150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol:water:formic acid (70:30:0.1, v/v/v). A tandem mass spectrometer equipped with electrospray ionization (ESI) source was used as the detector and operated in positive ion mode. The metabolite 602076 and the internal standard ZHY81018 were extracted from plasma by protein precipitation with acetonitrile. Chromatographic separation was performed on a Capcell MG C18 column (150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of acetonitrile and water (20:80, v/v). The MS/MS detection was operated in negative ion mode using an ESI source. The linear concentration ranges of the calibration curves were 2.5–500 ng/mL for ADF and 2.5–1000 ng/mL for 602076. The intra-assay RSD for quality control (QC) samples were from 3.3% to 6.7% for ADF, and 4.0% to 6.1% for 602076. The inter-assay RSD for QC samples were from 4.9% to 14.7% for ADF, and 2.6% to 4.4% for 602076. The relative errors for QC samples were from −10.6% to 1.9% for ADF, and 0.2% to 2.6% for 602076. The methods were successfully applied in the investigation of the pharmacokinetic profile of ADF, alamifovir and 602076 in rats. The results showed that ADF was rapidly metabolized to its active metabolite 602076 after oral absorption, with no detectable unchanged drug. The oral bioavailability of ADF was about 3 times higher than that of alamifovir.
Keywords: Alamifovir disoproxil fumarate; LC–MS/MS; Pharmacokinetics; Metabolite;

Bioavailability study of triamterene and xipamide using urinary pharmacokinetic data following single oral dose of each drug or their combination by Hadir M. Maher; Rasha M. Youssef; Eman I. El-Kimary; Ekram M. Hassan; Magda A. Barary (78-85).
► We develop HPLC–DAD methods for estimation of urinary triamterene and/or xipamide. ► Pharmacokinetic data for a single and combination oral doses were compared. ► Bioavailability of triamterene was increased from the combined formulation. ► Combination tablets are analyzed with a well-developed isocratic HPLC–DAD method.An efficient chromatographic method for the simultaneous determination of triamterene (TRI) and xipamide (XIP) in urine samples, based on high performance liquid chromatography with photodiode array detector (HPLC–DAD) has been developed. The HPLC separation was performed on a RP stainless-steel C-18 analytical column (250 mm × 4.6 mm, 5 μm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 4.0 and methanol as the mobile phase. The method was used to determine the urinary excretion profile and to calculate different urinary pharmacokinetic parameters following oral dose of their combination compared with single oral doses of each drug and hence comparing their bioavailability. Quantitation was performed using chlorthalidone as internal standard. The calibration graphs of each drug were rectilinear in the range of 0.2–40 μg/mL urine for TRI and 0.2–15 μg/mL urine for XIP. An HPLC–DAD method was also successfully developed for the simultaneous determination of the investigated drugs in pharmaceutical preparations. The methods were validated in terms of linearity, accuracy, precision, selectivity, limits of detection and quantitation and other aspects of analytical validation.
Keywords: Triamterene; Xipamide; Urine analysis; Pharmacokinetics; HPLC–DAD;

Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography–tandem mass spectrometry by Ling-Zhi Wang; Rina Yue-Ling Ong; Tan-Min Chin; Win-Lwin Thuya; Seow-Ching Wan; Andrea Li-Ann Wong; Sui-Yung Chan; Paul C. Ho; Boon-Cher Goh (86-92).
► Hydroxychloroquine (HCQ) can reverse the acquired resistance to gefitinib. ► Current HPLC-UV assay is tedious for determination of HCQ in blood. ► A rapid analytical method has been developed using LC–MS/MS. ► One run for quantification of HCQ in blood can be completed with only 3 min. ► The method has been successfully applied in human pharmacokinetics study of HCQ.A novel and specific liquid chromatography–tandem mass spectrometric method (LC–MS/MS) was developed and validated for the quantification of hydroxychloroquine in human blood using its stable labeled isotope, hydroxychloroquine-d4 as the internal standard. Chromatographic separation of analytes was achieved using an Agilent ZORBAX Eclipse XDB – C8 analytical HPLC column (50 mm × 2.1 mm, 5 μm). The mobile phase comprising water containing 0.1% formic acid–acetonitrile (94:6, v/v) was delivered isocratically at a flow rate of 0.5 mL/min. The column effluent was detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) and monitored by multiple reaction monitoring with positive mode. The precursor to product ion transitions of m/z 336 → 247 and m/z 340 → 251 were used to measure the analyte and IS, respectively. The assay demonstrated a good linear dynamic range of 5–2000 ng/mL for hydroxychloroquine in human blood, with coefficient of determination (r 2) of =0.9999. The values for intra-day and inter-day precisions of hydroxychloroquine were ≤7.86% with the accuracies ranged from 93.8% to 107.6%. The chromatographic run time was 3 min, making it possible to achieve a high throughput analysis. This method was used as a bio-analytical tool in a phase I clinical trial to quantify blood hydroxychloroquine concentrations in patients with non-small cell lung cancer receiving both hydroxychloroquine and gefitinib in their treatment.
Keywords: Hydroxychloroquine; Rapid; Quantification; LC–MS/MS; Human blood;

► MEPS for rapid piperazine extraction from urine samples. ► Good detection and quantitation limits using only 0.1 mL of sample and accessible analytical instrumentation. ► Total analysis time of 15 min per sample.A method using microextraction by packed sorbent (MEPS) and high performance liquid chromatography-diode array detection (HPLC-DAD) is described for the determination of piperazine-type stimulants in human urine. The studied compounds were 1-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(3-chlorophenyl) piperazine (mCPP) and 1-(4-methoxyphenyl) piperazine (MeOPP); 1-(2-chlorophenyl)-piperazine (oCPP) was used as internal standard (IS).The factors which might influence the extraction were screened previously using the fractional factorial design approach, and none of them influenced significantly the process.The procedure was linear for concentrations ranging from 0.1 (lower limit of quantitation – LLOQ) to 5 μg/mL, with determination coefficients (R 2) higher than 0.99 for all analytes in all runs. The limits of detection were 0.1 μg/mL for BZP and TFMPP, while for MeOPP and mCPP 0.05 μg/mL was obtained. Intra- and interday precision ranged from 1 to 14%, and accuracy was within a ±15% interval for all analytes, fulfilling the criteria normally accepted in bioanalytical method validation. Under the optimized conditions, extraction efficiency was higher than 80% for all analytes, except BZP (50%).MEPS showed to be a rapid (<2 min) and simple procedure for the determination of piperazine-type stimulants in human urine, allowing reducing the handling time and costs usually associated to this type of analysis. Furthermore, the fact that only 0.1 mL of sample is required make this method a valuable and powerful tool for drug monitoring in human urine in situations where those compounds are involved, for instance in forensic scenarios.
Keywords: Microextraction by packed sorbent; Piperazines; HPLC-DAD; Urine;

Assessment of the stereoselective fungal biotransformation of albendazole and its analysis by HPLC in polar organic mode by Viviane Cangerana Hilário; Daniel Blascke Carrão; Thiago Barth; Keyller Bastos Borges; Niege Araçari Jacometti Cardoso Furtado; Mônica Tallarico Pupo; Anderson Rodrigo Moraes de Oliveira (100-107).
A high-performance liquid chromatographic method using polar organic mode was developed to analyze albendazole (ABZ), albendazole sulfone (ABZSO2) and the chiral and active metabolite albendazole sulfoxide (ABZSOX, ricobendazole) that was further applied in stereoselective fungal biotransformation studies. The chromatographic separation was performed on a Chiralpak AS column using acetonitrile:ethanol (97:3, v/v) plus 0.2% triethylamine and 0.2% acetic acid as the mobile phase at a flow rate of 0.5 mL min−1. The present study employed hollow fiber liquid-phase microextraction as sample preparation. The method showed to be linear over the concentration range of 25–5000 ng mL−1 for each ABZSOX enantiomer, 200–10,000 ng mL−1 for ABZ and 50–1000 ng mL−1 for ABZSO2 metabolite with correlation coefficient (r) > 0.9934. The mean recoveries for ABZ, rac-ABZSOX and ABZSO2 were, respectively, 9%, 33% and 20% with relative standard deviation below 10%. Within-day and between-day precision and accuracy assays for these analytes were studied at three concentration levels and were lower than 15%. This study opens the door regarding the possibility of using fungi in obtaining of the active metabolite ricobendazole. Nigrospora sphaerica (Sacc.) E. W. Mason (SS67), Pestalotiopsis foedans (VR8), Papulaspora immersa Hotson (SS13) and Mucor rouxii were able to stereoselectively metabolize ABZ into its chiral metabolite. Among them, the fungus Mucor rouxii was the most efficient in the production of (+)-ABZSOX.
Keywords: Chiral analysis; Stereoselective fungal biotransformation; Polar organic mode; Albendazole; Ricobendazole;

Development of an UPLC–MS/MS method for the determination of antibiotic ertapenem on dried blood spots by Giancarlo la Marca; Elisa Giocaliere; Fabio Villanelli; Sabrina Malvagia; Silvia Funghini; Daniela Ombrone; Luca Filippi; Marina De Gaudio; Maurizio De Martino; Luisa Galli (108-113).
► We evaluate ertapenem concentration from DBS for the first time. ► Rapid chromatographic method on DBS in children for multiple analytes saves costs. ► The use of DBS for therapeutic drug monitoring in children is advantageous.Ertapenem (Invanz®) is a newly developed carbapenem β-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC–MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5–100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra- and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions.The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home.This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.
Keywords: Ertapenem; Carbapenem; DBS; UPLC–MS/MS;

Determining active pharmaceutical ingredient (API) tablet concentrations rapidly and efficiently is of great importance to the pharmaceutical industry in order to assure quality control. Using near-infrared (NIR) spectra measured on tablets in conjunction with multivariate calibration has been shown to meet these objectives. However, the calibration is typically developed under one set of conditions (primary conditions) and new tablets are produced under different measurement conditions (secondary conditions). Hence, the accuracy of multivariate calibration is limited due to differences between primary and secondary conditions such as tablet variances (composition, dosage, and production processes and precision), different instruments, and/or new environmental conditions. This study evaluates application of Tikhonov regularization (TR) to update NIR calibration models developed in a controlled primary laboratory setting to predict API tablet concentrations manufactured in full production where conditions and tablets are significantly different than in the laboratory. With just a few new tablets from full production, it is found that TR provides reduced prediction errors by as much as 64% in one situation compared to no model-updating. TR prediction errors are reduced by as much as 51% compared to local centering, another calibration maintenance method. The TR updated primary models are also found to predict as well as a full calibration model formed in the secondary conditions.
Keywords: Multivariate calibration; Tikhonov regularization; Calibration standardization and maintenance; Model-updating; NIR spectroscopy;

Synthesis, full chemical characterisation and development of validated methods for the quantification of the components found in the evolved “legal high” NRG-2 by Osama I.G. Khreit; Craig Irving; Elke Schmidt; John A. Parkinson; Niamh Nic Daeid; Oliver B. Sutcliffe (122-135).
The prevalence of these cathinone-derived “legal high” drugs has given rise to both legal and analytical challenges in the identification of these substances. Full chemical characterisation and robust HPLC methods for the determination of two new derivatives of mephedrone (namely 4-MEC and 4-MBC) identified in “street” samples of NRG-2 is presented.Display Omitted► Synthesis of two (±)-mephedrone derivatives: (±)-4′-methyl-N-ethylcathinone (4-MEC) and (±)-4′-methyl-N-benzylcathinone (4-MBC) identified in samples of NRG-2. ► Full and comprehensive analytical profiling of 4-MEC and 4-MBC. ► Fully validated chromatographic methods for the analysis of 4-MEC and 4-MBC in their pure form and in the presence of common adulterants. ► Evaluation of sourced samples of NRG-2.The recent global increase in the abuse of 4′-methylmethcathinone (mephedrone) and related compounds has developed a requirement for full chemical characterisation of these products. This work presents full synthetic and chemical characterisation data for the hydrobromide salts of two mephedrone derivatives: 4′-methyl-N-ethylcathinone (4-MEC) and 4′-methyl-N-benzylcathinone (4-MBC) which have been identified in samples of the “legal high” NRG-2. The first fully validated chromatographic methods for the detection and quantitative analysis of these substances both in their pure form and in the presence of a number of common adulterants used in illicit drug manufacture is also reported.
Keywords: NRG-2; 4-MEC; 4-MBC; Cathinones; HPLC;

Three commercial xenogeneic biomaterials (Gen-Os, Apatos Spongiosa and Apatos Cortical; all from Tecnoss Dental, Torino, Italy) originated from porcine bone were characterized by various analytical methods, such as powder X-ray diffraction (XRD), thermogravimetry (TGA), high-resolution solid-state nuclear magnetic resonance (ssNMR) and infrared spectroscopy (FT-IR). The studies were focused on structural properties and chemical compositions of the samples. It was found that the main constituents of the analyzed biomaterials were nanocrystalline apatite mineral, organic collagenous matrix and water. For comparison, synthetic carbonated hydroxyapatite and natural collagen type I from bovine tendon were used. Differences in various physicochemical parameters such as crystal size, specific surface area, concentration of structural hydroxyl groups, contents of CO3 2− and HPO4 2− ions and their location were discussed. It was shown that various techniques of ssNMR and elaborate analysis of the FT-IR spectra, applied together, provide valuable information on xenogeneic biomaterials.
Keywords: Bone substitute; Xenogeneic biomaterial; Biological apatite; Solid-state NMR; Infrared spectroscopy; Powder diffraction;

► We developed an UHPLC method that can separate corynanthine from yohimbine. ► Analysis of yohimbe bark and dietary supplements revealed quality problems. ► Quantitation of yohimbine by itself is not sufficient for dietary supplement quality control. ► Combination of quantitation and fingerprinting analysis is needed.A practical ultra high-performance liquid chromatography (UHPLC) method was developed for fingerprint analysis of and determination of yohimbine in yohimbe barks and related dietary supplements. Good separation was achieved using a Waters Acquity BEH C18 column with gradient elution using 0.1% (v/v) aqueous ammonium hydroxide and 0.1% ammonium hydroxide in methanol as the mobile phases. The study is the first reported chromatographic method that separates corynanthine from yohimbine in yohimbe bark extract. The chromatographic fingerprint analysis was applied to the analysis of 18 yohimbe commercial dietary supplement samples. Quantitation of yohimbine, the traditional method for analysis of yohimbe barks, were also performed to evaluate the results of the fingerprint analysis. Wide variability was observed in fingerprints and yohimbine content among yohimbe dietary supplement samples. For most of the dietary supplements, the yohimbine content was not consistent with the label claims.
Keywords: Fingerprint; UHPLC; Yohimbe; Yohimbine; Dietary supplements;

A new, simple and fit for purpose method based on liquid chromatography with UV spectrophotometric detection and post-column derivatisation (LC-UV-PCD) for the determination of semduramicin in poultry compound feed, premixtures and feed additive as well as its discrimination from other coccidiostats in poultry compound feed has been developed and single-laboratory validated. The concentration levels of the target analyte at which the validation experiments have been carried out varied between 12.8 and 51.3 mg kg−1 in compound feed, covering the authorised levels of semduramicin according to European Union legislation. Furthermore, the method has been validated for a premixture sample containing semduramicin at 3 g kg−1 and the feed additive containing semduramicin at 51 g kg−1. The method developed involved a simple extraction of the coccidiostats with acetonitrile from the feed samples followed by a filtration of the supernatants. The resulting supernatants were submitted to chromatographic analysis. When analysing the feed additive and the premixture samples, the extraction solution was appropriately diluted prior to LC-UV-PCD analysis. The analytes were quantified through an external calibration curve prepared with pure semduramicin standards. The relative standard deviations for repeatability and for intermediate precision varied from 2.4 to 8.8% and from 2.6 to 8.8%, respectively, and the values for the relative recovery rate ranged from 89 to 95%. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be below 1 mg kg−1 and 3 mg kg−1, respectively. Moreover, the results showed a comparable performance profile, when using methyl isobutyl ketone instead of acetonitrile as extraction solvent.Based on the obtained method performance characteristics, the method is considered suitable for the determination of semduramicin in poultry compound feed at authorised level, in premixtures and in the feed additive, hence allowing the enforcement of the European Union legislation regarding the control and the monitoring in feedingstuffs.
Keywords: Ionophore coccidiostats; Semduramicin; Feed; Single-laboratory validation; Ruggedness test;

The site-specific basicity of thyroid hormones and their precursors as regulators of their biological functions by Gergő Tóth; Sándor Hosztafi; Zsuzsanna Kovács; Béla Noszál (156-164).
► The protonation macro- and microconstants of thyroid hormones were determined. ► The results were interpreted in terms of biological functions. ► Protonation states of thyroid hormones are crucial for their biochemical functions.The complete macro- and microequilibrium analyses of thyroxine, liothyronine, reverse liothyronine and their biological precursors – diiodotyrosine, monoiodotyrosine and tyrosine are presented. Their biosyntheses, receptor- and transport protein-binding are shown to be distinctively dependent on the phenolate basicity. The protonation macroconstants were determined by 1H NMR-pH and/or UV-pH titrations. Microconstants of the minor microspecies were determined by deductive methods, in which O-methylated and carboxymethylated derivatives were synthesized, and the combination of their NMR-pH and UV-pH titration provided the experimental base to evaluate all the microconstants. NMR-pH profiles, macro-, and microscopic protonation schemes, and species-specific diagrams are included.Biosyntheses of the thyroid hormones take place by oxidative coupling of two iodotyrosine residues catalyzed by thyreoperoxidase in thyreoglobulin. On the grounds of our phenolate microconstants of precursors the thyroxine over liothyronine ratio needs to be 9:1 after their biosynthesis in thyroid gland, which is in good agreement with biochemical data. The microconstants show that the phenolates are in proton donor (–OH) form in liothyronine whereas they occur in proton acceptor (–O) form in thyroxine at the pH of blood. These facts explain several facts that have previously been empirically known: the affinity of liothyronine for the receptor is higher than that of thyroxine, the affinity of thyroxine for the transport proteins is higher than that of liothyronine and the selectivity of thyroxine for the OATP1C1 organic anion transporter is higher than that of liothyronine.
Keywords: NMR; Thyroxine; Protonation constants; Thyroid hormone biosynthesis; Microspeciation;

Highly efficient, selective, sensitive and stability indicating RP-HPLC–UV method for the quantitative determination of potential impurities and characterization of four novel impurities in eslicarbazepine acetate active pharmaceutical ingredient by LC/ESI-IT/MS/MS by Saji Thomas; Amber Bharti; Pawan Kumar Maddhesia; Sanjeev Shandilya; Ashutosh Agarwal; Dharamvir; Sujay Biswas; Vikas Bhansal; Ashish Kumar Gupta; Praveen Kumar Tewari; Chandra S. Mathela (165-175).
► First report on a stability indicating validated LC–UV method for the quantification of potential impurities. ► Four novel impurities were identified and characterized. ► Minimum detectable limits of impurities were found to be as low as 0.005%. ► New LC–UV method was found to be giving excellent resolution for 15 impurities.A novel, sensitive, selective and stability indicating LC–UV method was developed for the determination of potential impurities of eslicarbazepine acetate. High performance liquid chromatographic investigation of eslicarbazepine acetate laboratory sample revealed the presence of several impurities. Three impurities were characterized rapidly and four impurities were found to be unknown. The unknown impurities were identified by liquid chromatography coupled with electrospray ionization, ion trap mass spectrometry (LC/ESI-IT/MS/MS). Structural confirmation of these impurities was unambiguously carried out by synthesis followed by characterization using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (FT–IR) and mass spectrometry (MS). Based on the spectroscopic, spectrometric and elemental analysis data unknown impurities were characterized as 5-acetyl-5,11-dihydro-10H-dibenzo [b,f]azepin-10-one, N-acetyl-5H-dibenzo[b,f]azepine-5-carboxamide, 5-acetyl-10,11-dihydro-5H-dibenzo[b,f]azepin-10-yl acetate and 5-acetyl-5H-dibenzo[b,f]azepin-10-yl acetate. The newly developed LC–UV method was validated according to ICH guidelines considering eleven potential impurities and four new impurities to demonstrate specificity, precision, linearity, accuracy and stability indicating nature of the method. The newly developed method was found to be highly efficient, selective, sensitive and stability indicating. A plausible pathway for the formation of four new impurities is proposed.
Keywords: Eslicarbazepine acetate; Characterization; LC/MS/MS; Impurity; Validation;

Measurement of drug diffusivities in pharmaceutical solvents using Taylor dispersion analysis by Fengbin Ye; Henrik Jensen; Susan W. Larsen; Anan Yaghmur; Claus Larsen; Jesper Østergaard (176-183).
► Diffusivity was measured by Taylor dispersion using capillary with 2 detection points. ► Diffusivity measurements performed using two detection windows are more robust. ► The diffusivities of 11 compounds were in good agreement with the literature data. ► Simultaneous measurement ofdiffusivity and viscosity by Taylor dispersion analysis.Knowledge of drug diffusivity is of key importance in the understanding of a number of pharmaceutical and biological processes. However, experimentally determined diffusion coefficients and hydrodynamic radii are only reported for a limited number of drug substances. In this work, Taylor dispersion analysis conducted using capillary electrophoresis instrumentation coupled with a UV imaging detector, with two detection windows along the capillary, is introduced as a powerful method for the determination of drug diffusivities in nanoliter samples. Several potential advantages associated with applying two detection windows instead of one window as done in most previous studies were identified. Overall diffusion coefficient measurements performed using two detection windows are more robust and correction for changes in flow rate and sample volume is not required. The experimental conditions applied were suboptimal for performing single detection window measurements due to the relatively large sample volumes and may be optimized to alleviate the need for tedious correction procedures for this setup. The diffusivities of eleven aromatic compounds in water at 25 °C were determined, and showed a good agreement with the literature values. Furthermore, the diffusivities and hydrodynamic radii of four selected drug substances were determined in acetonitrile, methanol, isopropyl myristate, medium chain triglyceride, and propylene glycol in addition to water. The solvent viscosity was determined simultaneously along with the measurement of analyte diffusivity. Drug diffusivities decreased with increasing solvent viscosity. Taylor dispersion analysis is a robust, simple and automated method of quantification of diffusion coefficients even in media with a relatively higher viscosity than water.
Keywords: Diffusion coefficient; Hydrodynamic radius; Molecular size; Taylor dispersion analysis; Viscosity;

► Vesicle electrokinetic chromatography predicts the phospholipidosis-inducing potential of pharmaceuticals. ► Ion-exchange interaction relates to phospholipidosis-inducing potential. ► Volume distribution of drugs relates to its phospholipidosis-inducing potential.Drug-induced phospholipidosis (PLD) is an excessive accumulation of polar phospholipids within cells as a result of medical treatment. Even though a direct link between in vitro drug-induced PLD and toxicity in humans has not yet been firmly established, the development of PLD during preclinical testing in animals is a recognized problem in the pharmaceutical industry and can delay or abort the development process. Therefore, it is of interest to investigate the potential PLD risk of candidates at an early stage of the drug discovery process. In this work, a high throughput physicochemical approach, which is based on measuring the retention factors of the test compound within several different separation systems, was developed for screening phospholipidosis-inducing potential (PLIP) of pharmaceuticals. The measured retention factors of 36 drugs were compared with literature data on PLIP risk from three different sources. It is clearly shown that there is a statistical correlation between the chromatographic retention parameters of tested drugs and their PLIP risk. In conclusion, the retention factor (log  k AOT ) observed on a docusate sodium salt (AOT) surfactant vesicle electrokinetic chromatography (EKC) system and the logarithm of the volume of distribution (log  V d ) calculated from immobilized artificial membrane chromatography at pH 7.4 (CHI IAM7.4) and HSA binding value (% HSA) can provide primary profile prediction for a large number of drug candidates early in the drug discovery process with minimal resources. The observations are that the higher the value of both log  k AOT and log  V d , the higher the PLIP risk, and we would recommend this dual approach.
Keywords: Drug phospholipidosis-inducing potential; Volume of distribution; Phospholipidosis; Surfactant vesicle electrokinetic chromatography;

Libraries, classifiers, and quantifiers: A comparison of chemometric methods for the analysis of Raman spectra of contaminated pharmaceutical materials by Connie M. Gryniewicz-Ruzicka; Jason D. Rodriguez; Sergey Arzhantsev; Lucinda F. Buhse; John F. Kauffman (191-198).
► Raman method used to screen sorbitol for the presence of low level adulterants. ► Compared a Raman based library spectral correlation method to chemometric methods. ► Correlation methods cannot identify adulterants present in sorbitol below ∼10%. ► Classification methods flagged sorbitol samples when an adulterant >2% was present. ► Quantification by PLS had the lowest limit of detection for the adulterant at ∼0.9%.In this study, pharmaceutical grade sorbitol was used as a model system for comparison of Raman based library spectral correlation methods with more sophisticated methods of chemometric data analysis. Both crystallizing sorbitol (CS) and non-crystallizing sorbitol (NCS) from several manufacturers were examined. The Raman spectrum of each sample was collected and identified by correlation with a spectral library that included the CS spectrum but not the NCS spectrum. The average hit quality index (HQI) for the measured NCS spectra and the library CS spectrum was 0.966 whereas the average HQI for the measured CS spectra was 0.991. Both HQIs exceeded the 0.950 threshold that is commonly used for material verification. To enhance the discrimination between CS and NCS, a CS/NCS classification model was constructed using soft independent modeling of class analogies (SIMCA). SIMCA was able to positively identify CS and NCS solutions with no mis-classifications. When CS was adulterated with low levels (0–5%) of ethylene glycol (EG) and diethylene glycol (DEG), the HQI values of the measured spectra and the CS library spectrum were still above 0.950. When the CS SIMCA model was applied to adulterated CS spectra, it determined that CS samples with adulterant levels as low as 2% were outside of the CS class. A quantitative PLS model was also applied to EG adulterated CS and resulted in a detection limit of 0.9% for EG. The results obtained from these studies highlight the importance of selecting an appropriate data analysis process for the detection of low level adulterants in pharmaceutical raw materials using Raman spectroscopic screening methods.
Keywords: Rapid screening; Raman spectroscopy; Spectral library; SIMCA; PLS;

Preparative separation of dryofragin and aspidin BB from Dryopteris fragrans extracts by macroporous resin column chromatography by Xiao-Juan Li; Yu-Jie Fu; Meng Luo; Wei Wang; Lin Zhang; Chun-Jian Zhao; Yuan-Gang Zu (199-206).
► Application of macroporous resin column chromatography. ► Preparative separation of dryofragin and aspidin BB firstly. ► Multi-target products were enriched simultaneously.A simple, efficient and environment-friendly chromatographic separation method was developed for preparative separation and enrichment of dryofragin and aspidin BB from Dryopteris fragrans. The adsorption properties of twelve macroporous adsorption resins were evaluated. The three selected resins were further screened depending on the separation performance of their packed columns, in which AB-8 resin showed better separation efficiency for dryofragin and aspidin BB. In order to maximize column efficiency, the operating parameters (flow rate, ethanol concentration and volume) of the resin column chromatography were optimized and compared with the conventional resin column adsorption. After preparative separation and enrichment on resin column chromatography, the contents of dryofragin and aspidin BB in the product were 8.39- and 5.99-fold increased with recovery yields of 91.22% and 75.64%, respectively. Moreover, the regenerated adsorbent exhibited excellent reusability within at least five cycles of adsorption/desorption. It suggested that multi-targets would be enriched effectively by resin column chromatography.
Keywords: Aspidin BB; Column chromatography; Dryofragin; Macroporous resins; Separation;

Negundoside (1), agnuside (2) and 6′-p-hydroxy benzoyl mussaenosidic acid (3) are known bioactive metabolites in Vitex trifolia. In the present study a simple precise and reproducible method was developed for simultaneous quantitation of NS (1), AS (2) and HMA (3) and the antioxidant capacity of above markers has also been determined. Marker compounds have been resolved using silica gel 60 F254 plates, petroleum ether (60–80)/toluene/acetone/water (10:10:80:2 v/v/v/v) as the mobile phases. The method does not employ any derivatisation procedure and can be used as a quality control tool for routine analysis of drugs V. trifolia and V. negundo together with their commercial extracts. NS (1), AS (2) and HMA (3) showed significant activity in DPPH and NO radical scavenging assays.
Keywords: Vitex trifolia; Vitex negundo; HPTLC; Iridoids; Antioxidant;

Forced degradation study of thiocolchicoside: Characterization of its degradation products by Del Grosso Erika; Aprile Silvio; Grosa Giorgio (215-223).
► Hydrolytic, oxidative, and photolytic stresses of thiocolchicoside (TCC). ► Degradation products were detected and characterized using LC–MS n experiments. ► Five DPs were identified and confirmed by synthesis, MS, NMR and IR analyses. ► A comprehensive degradation scheme of TCC was proposed. ► D1SO and D3 are the indicators of TCC stability for oxidative and hydrolytic stress.Thiocolchicoside (TCC, N-[1,2-dimethoxy-10-methylsulphanyl-9-oxo-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydropyran-2-yloxy)-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-acetamide) was subjected to hydrolytic, oxidative, and photolytic stresses. TCC underwent degradation in acidic, basic, and oxidative conditions, while it was stable toward other stress conditions. The degradation products (DPs) were detected and their separation was achieved on a SGE Wakosil C18RS 5 μm (250 * 4.6 mm; SGE) column employing a gradient LC–MS method for a total time of analysis of 18 min. The mass fragmentation pathways of both thiocolchicoside and its degradation products were established using LC–MS experiments assigning the structures to the DPs. In particular, five DPs were identified as: D1SO (N-[1,2-dimethoxy-10-methylsulphoxide-9-oxo-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydropyran-2-yloxy)-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-acetamide), D1SO 2 (N-[1,2-dimethoxy-10-methylsulphone-9-oxo-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydropyran-2-yloxy)-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-acetamide), D2 ([1,2-dimethoxy-10-methylsulphanyl-9-oxo-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydropyran-2-yloxy)-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-amine), D3 (N-[1,2-dimethoxy-3-hydroxy-10-methylsulphanyl-9-oxo-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-acetamide or 3-O-demethylthiocolchicine), D4 ([1,2-dimethoxy-3-hydroxy-10-methylsulphanyl-9-oxo-5,6,7,9-tetrahydro-benzo[a]heptalen-7-yl]-amine or N-deacetyl-3-O-demethylthiocochicine). Moreover, the structures of DPs were confirmed by synthesis of the reference standards which were fully characterized by MS, NMR, IR analyses. Finally a comprehensive degradation scheme of TCC was proposed allowing to outline D1SO and D3 as the indicators of its stability for oxidative and hydrolytic stress conditions.
Keywords: Thiocolchicoside; Forced degradation; Degradation products; LC–MS; LC–MS n ;

A method for identifying the origin of chondroitin sulfate with near infrared spectroscopy by Hengchang Zang; Lian Li; Fengshan Wang; Qiong Yi; Qin Dong; Chunxiao Sun; Jinfeng Wang (224-229).
The object of this study was to explore the feasibility of support vector machine (SVM) to identify the origin of chondroitin sulfate (CS) by near infrared spectroscopy. 96 batches CS from three different origins were collected in this research, 66 batches of which were chosen for training set by Kennard–Stone (KS) method and the rest were used for testing. Through the comparison of pretreatment methods of standard normal variate transformation (SNV), multiplicative scatter correction (MSC), Savitzky-Golay (SG) smoothing and derivative with SG smoothing, a SVM discrimination model was constructed, of which the prediction result is 100% accurate with the pretreatment of first derivative and SG smoothing with 15 points. The result indicated that it had great potential using SVM to identify the origin of CS.
Keywords: Chondroitin sulfate; Near infrared spectroscopy; Origin identification; SVM;

Development of chromatographic methods for the determination of genotoxic impurities in cloperastine fendizoate by Antonia García; Francisco J. Rupérez; Florencia Ceppa; Federica Pellati; Coral Barbas (230-236).
► Determination of genotoxic impurities with different physico-chemical properties in cloperastine phendizoate at ppb level.The classification of an impurity of a drug substance as genotoxic means that the “threshold of toxicological concern” (TTC) value of 1.5 μg/day intake, considered to be associated with an acceptable risk, should be the admissible limit in the raw material and that leads to new analytical challenges. In this study, reliable chromatographic methods were developed and applied as limit tests for the control of three genotoxic impurities (GTIs) in cloperastine fendizoate, drug widely used as an antitussive active pharmaceutical ingredient (API). In particular, GC–MS was applied to the determination of one alkyl halide (2-chloroethanol, 2-CE), while HPLC-DAD was selected for the analysis of two sulfonate esters (methyl p-toluenesulfonate, MPTS, and 2-chloroethyl p-toluenesulfonate, CEPTS).Regarding GC–MS, strong anion-exchange (SAX)-SPE was applied to remove fendizoate from the sample solutions, due its low volatility and its high amount in the raw material. The GC–MS analysis was performed on a Factor Four VF-23ms capillary column (30 m × 0.25 mm I.D., film thickness 0.25 μm, Varian). Single ion-monitoring (SIM) detection mode was set at m/z 80.In the case of HPLC-DAD, a suitable optimization of the chromatographic conditions was carried out in order to obtain a good separation of the impurity peaks from the drug substance peaks. The optimized method utilizes a SymmetryShield RP8 column (250 mm × 4.6 mm, 5 μm, Waters) kept at 50 °C, with phosphate buffer (pH 3.0; 10 mM)–methanol (containing 10% ACN) (45:55, v/v) as the mobile phase, at the flow-rate of 1.7 mL/min and UV detection at 227 nm. The required sensitivity level was achieved by injecting 80 μL of sample solution, purified from fendizoate by SAX-SPE, followed by a 1:1 (v/v) dilution of the SPE eluate with water.For both GC–MS and HPLC-DAD, the method validation was performed in relation to specificity and limit of detection (LOD), as required by ICH guidelines in relation to limit assays. The developed methods were successfully applied for the determination of GTIs in five different batches of cloperastine fendizoate. In all the analyzed batches, the three target GTIs were below the concentration limit.
Keywords: Cloperastine fendizoate; Genotoxic impurities; Sulfonate esters; Alkyl halides; Threshold of toxicological concern; Limit test; HPLC; GC;

Determination of cinacalcet hydrochloride in human plasma by liquid chromatography–tandem mass spectrometry by Fen Yang; Hongyun Wang; Qian Zhao; Hongzhong Liu; Pei Hu; Ji Jiang (237-241).
► An assay for the determination of KRN1493 using LC–MS/MS in MRM mode was developed. ► The method required small plasma volume and presented high throughput analysis. ► The present method was validated according to FDA guideline. ► This method was applied to the PK studies of KRN1493 in Chinese healthy volunteers.A sensitive and selective high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to determine the cinacalcet hydrochloride in human plasma. The analyte was extracted from plasma samples using a 96-well plate automatic solid-phase extraction (SPE) device and chromatographed on an Inertsil SIL-150 (2.1 mm × 50 mm, i.d. 5 μm) column using acetonitrile–water–formic acid (90:10:1) as the mobile phase with an isocratic flow rate of 0.35 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.1–25 ng/mL. The indicators of inter- and intra-day precision (RSD%) were all within 15.1%, and the accuracy (RE%) was within ±15%. The lower limit of quantitation (LLOQ) was 0.1 ng/mL. The average extraction recovery was 51.7%, and the detection was not affected by the matrix. The method was successfully applied to a pharmacokinetic study of cinacalcet hydrochloride in healthy Chinese volunteers.
Keywords: Cinacalcet hydrochloride; Calcimimetic agent; SPE; LC–MS/MS; MRM;

Quantitative determination and pharmacokinetic study of casticin in rat plasma by liquid chromatography–mass spectrometry by Jinlong Xu; Qiaoyan Zhang; Liang Zhao; Ying Wang; Liming Xue; Ting Han; Chengjian Zheng; Luping Qin (242-246).
A specific and sensitive liquid chromatography–mass spectrometry method has been developed and validated for identification and quantification of casticin in rat plasma after oral and intravenous administrations. Kaempferol was employed as an internal standard (IS). Liquid–liquid extraction using dichloromethane was applied to extract the casticin and the internal standard from plasma. Chromatographic separation was achieved on a Zorbax SB C18 column (100 mm × 3.0 mm, i.d.: 3.5 μm) with a mobile phase of methanol: 0.05% formic acid aqueous solution (60:40, v/v) at a flow rate of 0.4 mL/min for 10 min. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of detection (LLOD) was 7 ng/mL (S/N  = 5). The calibration curves were linear (r  > 0.9996) over the concentration range from 14.06 to 7187 ng/mL. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −8.2% to 1.0%. The extraction recovery at three quality control (QC) concentrations ranged from 91% to 102%. The validated method was successfully applied to the pharmacokinetic study of casticin after both an oral and an intravenous administrations to rats and the absolute bioavailability is 45.5 ± 11.0%.
Keywords: Casticin; Pharmacokinetics; Liquid chromatography; Mass spectrometry;

► Four quercetin glycosides were identified and quantified in Sussurea grandifolia. ► An uncommon quercetin glycoside, saxifragin (Qc-5-glc) was isolated from that plant. ► One gram of the MeOH extract contains 33.74 mg of saxifragin. ► That compound has also a potent peroxynitrite-scavenging activity (IC50, 0.67 μM).The leaves of Saussurea grandifolia (Compositae) are used as chwinamul, a well-known Korean mountainous vegetable, or to treat hepatitis and hematuria. Since the methanolic extract of S. grandifolia leaves exhibit a potent peroxynitrite-scavenging effect, phytochemical and high-performance liquid chromatographic (HPLC) analyses were employed to identify and simultaneously quantify the active components: chlorogenic acid and 3,5-di-O-caffeoylquinic acid (3,5-DQ) as caffeoylquinic acids, and quercetin, isoquercitrin (quercetin-3-glucoside), saxifragin (quercetin-5-glucoside), and rutin (quercetin-3-rutinoside). Validation of HPLC methods was performed to verify the linearity, LOD, LOQ, intra-day and inter-day variabilities, recovery, and repeatability to ensure that this method is selective, sensitive, precise, accurate and reproducible. In particular, leaves contained saxifragin with potent peroxynitrite-scavenging activity (IC50, 0.67 μM) as 4.65 mg/g dry weight, which is equivalent to 33.74 mg/g extract.
Keywords: Saussurea grandifolia; Compositae; Saxifragin; Peroxynitrite; HPLC;

► Selective and sensitive method of liquid chromatography-electrochemical detection. ► Analysis of active components of tetrandrine and fangchinoline in Chinese herb. ► High sensitive with limit of detection 270 nM. ► Less interference from complex matrix in real sample. ► LC–MS identification and confirmation.A simple and selective method was developed for the simultaneous determination of tetrandrine and fangchinoline in herbal medicine by HPLC with electrochemical detection (ECD) on a bare glassy carbon electrode. The HPLC separation and ECD conditions have been optimized. The separation was carried out on a WondaSil® C18-WR column (4.6 mm × 250 mm, 5 μm), with the mobile phase of acetonitrile–ammonium acetate buffer (pH 6.5; 40 mM) (32:68, v/v) using an isocratic elution at the flow rate of 0.5 mL/min. The electrochemical detection potential was set at +0.9 V. The obtained LODs for tetrandrine and fangchinoline were 0.26 and 0.27 μmol/L, respectively. The method was successfully applied to the analysis of tetrandrine and fangchinoline contents in Stephania tetrandra S. Moore. It has been demonstrated that the LC-ECD method is an excellent technique for analysis of the herbal medicine. The mean recoveries were in the range of 95–105%, while the precision expressed as repetition of peak area was lower than 2.7%.
Keywords: Tetrandrine; Fangchinoline; Stephania tetrandra S. Moore; LC-ECD; Herbal medicine;

Isolation and characterisation of degradant impurities in Dipyridamole formulation by B. Venkata Subbaiah; K.K. Sree Ganesh; G. Vamsi krishna; K. Vyas; R. Vasu Dev; K. Subramanyam Reddy (256-264).
Dipyridamole is an antithrombotic drug. In the stability study of drug product of Dipyridamole, two unknown impurities (referred as DP-I and DP-II) were detected at levels of 0.25% and 0.54% by gradient reverse phase HPLC method. The drug product was subjected to stress to enhance the level of these impurities. An elegant isocratic preparative method was employed using a Reprosil CN column with a short run time of 14 min to isolate these impurities. The DP-I and DP-II were isolated with purities of 99.1% and 99.8% respectively. Structural studies of these impurities were undertaken using spectroscopic techniques such as IR, NMR and Mass. Based on the spectral data, the structures of DP-I and DP-II have been characterised to be 2,2′,2″,2′″-(4-hydroxy-8-(piperidin-1-yl) pyrimido [5,4-d]pyrimidine-2,6 diyl) bis(azanetriyl) tetraethanol, 4-(2-((6-(bis (2-hydroxyethyl) amino)-4, 8-di (piperidin-1-yl) pyrimido [5,4-d] pyrimidin-2-yl) (2-hydroxyethyl) amino) ethoxy)-2, 3-dihydroxy-4-oxobutanoic acid, respectively. A detailed elucidation of the structure is presented in this article.
Keywords: Dipyridamole; Degradant impurities; Preparative HPLC; Isolation; Characterisation techniques;

A novel HPTLC-densitometry method was developed for separation and quantitative determination of four flavonoids: quercetin 3-O-β-d-glucuropyranoside (miquelianin; QG), quercetin 3-O-β-d-glucopyranoside (isoquercitrin; IQ), quercetin 3-O-β-d-galactopyranoside (hyperoside; HYP) and quercetin 3-O-β-d-(6″-α-l-rhamnosylo)-glucopyranoside (rutin; RUT) in ethyl acetate fractions from aerial parts of selected Potentilla species: P. argentea, P. erecta, Dasiphora fruticosa (syn. P. fruticosa), Drymocallis rupestris (syn. P. rupestris), P. nepalensis var. ‘Miss Wilmott’ and P. thuringiaca. For the first time, separation of this type of flavonoids was achieved on a HPTLC DIOL F254 plates using a mixture consisting of ethyl acetate/methyl ethyl ketone/diisopropyl ether/formic acid (3:10:4:1, v/v/v/v). QG, IQ, HYP and RUT were determined by densitometry at 363 nm. Sensitivity, accuracy (recovery rates were between 95.0 and 101.4%) and precision (in both cases intra-day precision and inter-day precision were ≤8.0%) of the method were determined. Their amounts were calculated using the regression equations of the calibration curves which were linear in a range of 0.025–0.200 μg/spot for all investigated compounds. The amounts of marker compounds in ethyl acetate extracts of Potentilla species measured by the method ranged between 16.7 ± 1.1 and 41.7 ± 0.6 mg/g for QG, 15.8 ± 1.3 and 36.7 ± 1.0 mg/g for IQ, 14.5 ± 0.5 mg/g for HYP and 6.7 ± 0.3 and 27.8 ± 2.1 mg/g for RUT. The method was found to be relatively simple, specific, precise and accurate and may be used for the quality control of simultaneous determination of quercetin derivatives in Potentilla extracts but also in other similar plant materials.
Keywords: Potentilla L.; High-performance thin-layer chromatography (HPTLC); Densitometry; Flavonoids; Quercetin derivatives;

► Development and validation of new liquid chromatography (LC) method with UV detection for the simultaneous determination of clopidogrel (CLP) and acetylsalicylic acid (ASA) and their related substances in combined oral formulations. ► Column and mobile phase selection. ► Optimization of the LC-UV method – chromatographic conditions. ► Separation of CLP and ASA from their related substances. ► Application of the developed LC-UV method in three dosage forms – quantitative determination of CLP and ASA and their related substances.A reversed phase liquid chromatographic method with UV detection for the simultaneous determination of clopidogrel and acetylsalicylic acid and their related substances in combined oral formulations was developed and validated. Good separation was achieved on a Luna C18 column (150 mm × 4.6 mm, 3 μm) using gradient elution at a flow rate of 1 mL/min and a column temperature of 35 °C. UV detection was performed at 220 nm. The validation was performed according to the ICH guidelines. The method proved to be specific, sensitive (LOQ = 0.975 μg/mL and 0.0384 μg/mL for clopidogrel and acetylsalicylic acid, respectively), linear in the concentration range from LOQ to 325 μg/mL for clopidogrel and from LOQ to 650 μg/mL for acetylsalicylic acid, precise (RSD values for intermediate precision <1%) and accurate with mean recovery values of 100.7% and 100.2% for clopidogrel and acetylsalicylic acid, respectively. Moreover, the solution stability and method robustness were examined. The method gives satisfactory separation of impurities of clopidogrel and acetylsalicylic acid and so it is suitable for quantification of the related substances as well as for the assay of the actives.
Keywords: Aspirin; Clopidogrel; Reversed phase liquid chromatography; Impurities; Method development;

Mechanistic study on degradation of azelnidipine solution under radical initiator-based oxidative conditions by Eiji Ueyama; Fumie Takahashi; Jyunji Ohashi; Tomonori Konse; Naoyuki Kishi; Kenji Kano (277-283).
► A stability-indicating method that can realize the separation of all of the oxidative degradates of azelnidipine has been developed. ► The structure of the four major degradates of AZ generated under AIBN-initiated oxidation conditions has been characterized, and the mechanistic pathways have been proposed. ► An oxidation experiment with heavy oxygen water as an oxygen tracer has proved water molecule participation in the Dg-B generation step.We identified four degradants (Dg-A, Dg-B, Dg-C, Dg-D) of azelnidipine to be generated under radical initiator-based oxidative conditions and proposed the mechanistic pathway for their formation. 2,2′-Azobisisobutyronitrile was used as a radical initiator. There appeared to be two major pathways in the oxidation of the 1,4-dihydropyridine moiety. One was initiated by hydrogen abstraction from the C-4 position of the dihydropyridine ring, followed by hydrogen abstraction from the N-1 position, leading to aromatization of the dihydropyridine ring and Dg-A generation. The other was initiated by hydrogen abstraction from the N-1 position of the dihydropyridine ring followed by oxidation and hydrolysis to yield Dg-B. Furthermore, Dg-B was subjected to hydrolysis to generate Dg-C and Dg-D. It has been revealed that the rate of the Dg-B degradation was predominantly governed by the water content of the solvent used. Water participation in Dg-B degradation was proved by monitoring the incorporation of heavy oxygen atom (18O) into the structure with LC–MS, in which the experiment was carried out in a medium prepared with heavy oxygen water to label 18O during the hydrolysis.
Keywords: AIBN; Oxidation; Stability; Heavy oxygen water; Degradation product;