Journal of Chromatography B (v.1027, #C)

Vosaroxin is a first-in-class anticancer quinolone derivative topoisomerase II inhibitor that is currently in development in combination with cytarabine for the treatment of acute myeloid leukemia (AML). To investigate vosaroxin pharmacokinetics (PK) in patients, liquid chromatography tandem mass spectrometry (LC–MS/MS) assays to quantify vosaroxin and the two metabolites N-desmethylvosaroxin and O-desmethylvosaroxin in human plasma and urine were developed and validated.Immediately after collection the samples were stored at −80 °C. Prior to analysis, the plasma samples were subjected to protein precipitation and the urine samples were diluted. For both assays the reconstituted extracts were injected on a Symmetry Shield RP8 column and gradient elution was applied using 0.1% formic acid in water and acetonitrile-methanol (50:50, v/v). Analyses were performed with a triple quadruple mass spectrometer in positive-ion mode. A deuterated isotope of vosaroxin was used as internal standard for the quantification.The validated assays quantify vosaroxin and N-desmethylvosaroxin in the concentration range of 2–500 ng/mL in plasma and urine. For O-desmethylvosaroxin the concentration range of 4–500 ng/mL in plasma and urine was validated. Dilution integrity experiments show that samples can be diluted 25 fold in control matrix prior to analysis. The expanded concentration range for plasma and urine for vosaroxin and N-desmethylvosaroxin is therefore from 2 to 15,000 ng/mL and in plasma for O-desmethylvosaroxin from 4 to 15,000 ng/mL.
Keywords: Vosaroxin; N-desmethylvosaroxin; O-desmethylvosaroxin; HPLC–MS/MS; GLP; Pharmacokinetics; Clinical pharmacology;

We present a UPLC®-High Resolution Mass Spectrometric method to simultaneously screen for nineteen benzodiazepines, twelve opiates, cocaine and three metabolites, and three “Z-drug” hypnotic sedatives in both blood and urine specimens. Sample processing consists of a high-speed, high-temperature enzymatic hydrolysis for urine samples followed by a rapid supported liquid extraction (SLE). The combination of ultra-high resolution chromatography with high resolution mass spectrometry allows all 38 analytes to be uniquely detected with a ten minute analytical run. Limits of detection for all target analytes are 3 ng/mL or better, with only 0.3 mL of specimen used for analysis. The combination of low sample volume with fast processing and analysis makes this method a suitable replacement for immunoassay screening of the targeted drug classes, while providing far superior specificity and better limits of detection than can routinely be obtained by immunoassay.
Keywords: Drugs of abuse; Screening procedure; Orbitrap; UPLC®; LC–MS;

Determination of 1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in human serum using liquid chromatography with tandem mass spectrometry by Huiling Fang; Songlin Yu; Qian Cheng; Xinqi Cheng; Jianhua Han; Xuzhen Qin; Liangyu Xia; Xiaomei Jiang; Ling Qiu (19-26).
Vitamin D plays important roles in skeletal metabolism and many other diseases, including chronic renal failure, hypoparathyroidism, sarcoidosis and rickets. 1α,25-dihydroxy vitamin D (1α,25(OH)2D), the active form of vitamin D, exhibits an extremely low serum concentration, which makes its quantification very challenging. High performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) is considered to be the “gold standard” for the determination of these chemicals, which are found in low concentrations in the serum, but conventionally, it needs tedious sample pretreatment procedures, such as solid phase extraction and derivatization. Herein, we describe a simple and rapid HPLC–MS/MS method for the simultaneous quantification of 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and 1α,25-dihydroxy vitamin D2 (1α,25(OH)2D2). The analytes were extracted from the matrix by liquid–liquid extraction, centrifuged to dryness and reconstituted with 75% methanol. Lithium acetate was employed to improve the ionization efficiency of 1α,25(OH)2D. The assay was sensitive with a low limit of quantitation of 10.0 pg/mL for both 1α,25(OH)2D3 and 1α,25(OH)2D2 using a 0.5 mL sample aliquot. Linearity was obtained over the range of 10.0 pg/mL to 500 pg/mL. Both the inter-assay and intra-assay precisions were <15%, and the analytical recoveries were within 100 ± 5%. The performance evaluation of this assay demonstrated that it was a practical, sensitive and specific method for measuring the serum 1α,25(OH)2D3 and 1α,25(OH)2D2 concentrations.
Keywords: 1,25-Dihydroxy vitamin D3; 1,25-Dihydroxy vitamin D2; LC–MS/MS; Serum;

Both qualitative and quantitative analyses combined with activity evaluation were used for the integrated quality control study of Pien-Tze-Huang (PZH), a precious lozenge used for 460 years in China. In qualitative analysis, a high performance LC-quadrupole time-of-flight MS method was developed for the identification of chemical profiling in PZH. 27 compounds were unambiguously identified by comparing with reference substances, and 12 compounds were tentatively deduced based on their MS data and comparing with literatures. Then the in vitro anti-inflammatory activity of 27 compounds were evaluated, and 9 saponins, 11 bile acids, taurine and muscone exhibited significant inhibitory activities on TNF-α production with IC50 values ranging from 12.34 to 147.24 μM. In quantitative analysis, 21 active compounds, selected as quality control markers, were simultaneously detected in 15 batches of PZH samples by ultra-performance LC-triple quadrupole MS. All the analytes were detected within 15 min. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r 2  > 0.9990). The respective relative standard deviations of intra- and inter-day precisions, repeatability, stability, and recovery were less than 4.32%, 4.94%, 4.78%, 4.87%, and 4.91%. This study established a high sensitive and efficient method for the integrated quality control of PZH, including qualitative and quantitative analyses combined with pharmacological activity evaluation.
Keywords: Pian-Tze-Huang; Identification and quantification; Quality control; Mass spectrometry; Anti-inflammatory activity;

Metabolomics is an “omic” technique being increasingly used in epidemiological and clinical studies. We developed a method combining untargeted metabolomics with the quantitative determination of eight amino acids (AA) and eight acylcarnitines (AC) in plasma using ultra-high pressure liquid chromatography (UHPLC), electrospray ionization (ESI) and quadrupole time-of-flight mass spectrometry (QTOFMS). Separation of metabolites is performed by ion-pair reverse phase UHPLC using a HSS T3 column (2.1 × 100 mm, 100 Å, 1.8 μm particle size) and formic acid–ammonium acetate-heptafluorobutyric acid in water and formic acid–ammonium acetate in methanol as mobile phases. Metabolite identification and quantification are achieved using a QTOFMS operating in ESI-positive and full-scan mode along with MSE acquisition of fragmentation patterns. Targeted metabolites are quantified using the appropriate labeled standards and include branched-chain AA (leucine, isoleucine, valine), aromatic AA (phenylalanine, tyrosine) as well as acetylcarnitine and propionylcarnitine, which have been identified as biomarkers of future cardiometabolic disease risk. The inter-day precision (relative standard deviation) for the targeted method was <15% for all but one metabolite and accuracy (bias) of amino acids ranged from 0.5% to 13.9% using SRM 1950 as the external standard. Untargeted metabolomics in 30 plasma samples from the general Canadian population revealed 5018 features, of which 48 metabolites were identified using the MZmine 2.19 software including 23 by our in-house library that comprises 671 annotated metabolites. SRM 1950 analysis revealed 11,684 features, among which 154 metabolites were identified. Our method is currently applied in several epidemiological studies to better characterize cardiometabolic diseases and identify new biomarkers for disease prevention.
Keywords: Amino acids; Acylcarnitines; Metabolomics; Metabolites; Method validation; Plasma; UHPLC-ESI-QTOFMS;

Electro-deposited poly-luminol molecularly imprinted polymer coating on carboxyl graphene for stir bar sorptive extraction of estrogens in milk by Haibo Liu; Li Qiao; Ning Gan; Saichai Lin; Yuting Cao; Futao Hu; Jiayu Wang; Yinji Chen (50-56).
Electrochemical polymerization of luminol molecularly imprinted polymer on carboxyl graphene (MIP/CG) was developed as stir bar sorptive extraction (SBSE) coating for selective pre-concentration and specific recognition of bisphenol A (BPA), hexoestrol and diethylstilbestrol in milk samples. Luminol was employed as monomer and BPA as the template to prepare MIP under 0–0.6 V electro-polymerization. Carboxyl graphene was modified on pencil lead as the substrate to increase extraction capacity. The preparation and extraction conditions affecting the extraction efficiency were optimized. Under the optimized conditions, a good linearity of three estrogens was obtained in the range of 4–1000 ng mL−1. The average recoveries at the three spiked levels of the three estrogens ranged from 83.4% to 96.3% with the relative standard deviations (RSD) ≤ 7.1%. The limits of detection were in the range of 0.36–1.09 ng mL−1. The developed method with low cost, high selectivity and good reproducibility can be potentially applied for determining trace estrogens in complex food samples.
Keywords: Electrochemical polymerization; Polyluminol; Molecularly imprinted polymer; Stir bar sorptive extraction; Estrogens;

Chromatographic resolution of angiotensin II receptor antagonists (sartans) by Muhammad Saqlain Tahir; Ahmad Adnan; Quratulain Syed (57-63).
First time a simple, sensitive and unified quantification method has been developed to analyze the complete class of angiotensin II receptor antagonists which are used in the treatment of hypertension either alone or in combination with some other drugs. The most important advantage of developed method was that the eight separate drugs can be determined on a single chromatographic system without modifications in detection wavelength and mobile phase. The drugs were separated on a Purospher Star 4.6 mm × 25 cm, 5 μm, C18 column maintained at 40 °C with 1 mL min−1 flow rate using ultra violet detection at 254 nm. Good separation (Rs > 2.0) was achieved in a short analysis allowing simultaneous determination of all eight sartans. The effect of variation in flow rate, detection wavelength and column oven temperature was also studied. The proposed method was statistically validated in terms of precision, accuracy, linearity, specificity and robustness. The newly developed method proved to be specific, robust and accurate for the quantification of eight sartans in commercial pharmaceutical formulations.
Keywords: HPLC; Drug analogues; Sartans; Unified quantification; Validation;

Lycopus lucidus Turcz has been used as a kind of edible and medicinal material in eastern Asian countries. It has various bioactivities, including treatment of menstrual disorder, amenorrhea, menstrual cramps, inflammation, and cardiovascular diseases. However, the in vivo metabolism of L. lucidus Turcz extract is still not well described. In this study, L. lucidus Turcz extracts were administered to rats. Urine and fecal samples were collected at the difference periods (0–12 h, 12–24 h, and 24–36 h). Ultra-high performance liquid chromatography coupled with electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UHPLC−Q-TOF-MS) method was developed to characterize and identify the metabolites. A total of 17 metabolites in feces and 19 metabolites in urine were tentatively identified by means of accurate mass and characteristic fragment ions. The results show that glucuronidation and sulfation are the major metabolic reactions. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways of bioactive components might provide further understanding of the metabolic fate of the chemical constituents after oral administration of L. lucidus Turcz extract in rats.
Keywords: Lycopus lucidus Turcz; UHPLC−Q-TOF-MS; Metabolites; Identification;

A new method for the simultaneous determination of the three antifungal drugs using ultrasonic-assisted supramolecular dispersive liquid–liquid microextraction based on solidification of a floating organic droplet (UASMDLLME-SFO) was proposed. The supramolecular solvents produced from reversed micelles of 1-dodecanol (extraction solvent) in tetrahydrofuran (THF) were injected into the aqueous sample solution. Reverse micelle coacervates were produced in situ through self-assembly processes. The antifungal drugs were extracted from the aqueous sample into a supramolecular solvent. Sonication accelerated the mass transfer of the target analytes into the supramolecular solvent phase and enhanced the dispersion process. Some parameters affecting the extraction efficiency such as type and volume of the extraction solvent, pH, volume of the disperser solvent and ultrasound extraction time were investigated. Under optimum conditions, the limits of detections for ketoconazole, clotrimazole and miconazole ranged from 0.08 to 1.3 μgL−1 and the relative standard deviations (RSDs, n = 5) < 6% were obtained. The method was successfully applied for preconcentration of the three drugs in biological and water samples.
Keywords: Antifungal drugs; Dispersive liquid–liquid microextraction; Supramolecular; Solidification; Sonication;

Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10 by Nurul Lyana Md Sidek; Joo Shun Tan; Sahar Abbasiliasi; Fadzlie Wong Faizal Wong; Shuhaimi Mustafa; Arbakariya B. Ariff (81-87).
An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30 min and FR of 20 mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10–15 kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.
Keywords: Bacteriocin-like inhibitory substance; Pediococcus acidilactici Kp10; Purification; Aqueous two-phase flotation; Polyethylene glycol;

Sensitive electrophoretic method for determination of total sodium 2-mercaptoethanesulfonate (mesna) in human plasma, based on the stacking with high salt concentration in MEKC and in-capillary derivatization with 2-chloro-1-methyllepidinium tetrafluoroborate followed by UV detection was developed. In the method 0.03 mol L−1  pH 7 phosphate buffer with the addition of 0.01 mol L−1 SDS, and 10% ACN was used as a BGE. The limit of quantification (LOQ) of the method was 0.5 μmol L−1. Linearity in detector response was observed over the range of 0.5–10 μmol L−1 with the correlation coefficient 0.9971. The intra- and inter-day accuracy (three concentration levels, 5 days, n = 3) of the method ranged from 97.2 to 110.0% and from 94.0 to 101.2%, respectively. The novel MEKC method with UV detection proved to be suitable for determination of total mesna in human plasma.
Keywords: 2-Chloro-1-methyllepidinium tetrafluoroborate; In-capillary derivatization; Mesna; Plasma; Stacking with high salt concentration in MEKC;

Optimized ultra performance liquid chromatography tandem high resolution mass spectrometry method for the quantification of paraquat in plasma and urine by Haihua Lu; Jing Yu; Linlin Wu; Jingjing Xing; Jun Wang; Peipei Huang; Jinsong Zhang; Hang Xiao; Rong Gao (96-102).
A simple, sensitive and specific ultra performance liquid chromatography coupled to electrospray tandem high resolution mass spectrometry (UPLC-ESI-HRMS/MS) method has been developed and validated for quantification of paraquat in plasma and urine. The sample preparation was carried out by one-step protein precipitation with acetonitrile. The paraquat was separated with a HILIC column in 10 min. Detection was performed using Q Exactive Orbitrap mass spectrometer by Targeted-MS/MS scan mode. Methodological parameters, such as ammonium formate concentration, formic acid concentration, spray voltage, capillary temperature, heater temperature and normalized collision energy were optimized to achieve the highest sensitivity. The calibration curve was linear over the concentration range of LOQ-1000 ng/mL. LOD was 0.1 and 0.3 ng/mL, LOQ was 0.3 and 0.8 ng/mL for urine and plasma, respectively. The intra- and inter-day precisions were <7.97% and 4.78% for plasma and urine. The accuracies were within the range 93.51-100.90%. The plasma and urine matrices had negligible relative matrix effect in this study. This method was successfully applied to determine paraquat concentration in plasma samples with hemoperfusion from 5 suspected paraquat poisoning patients.
Keywords: Paraquat; HILIC column; UPLC-ESI-HRMS/MS; Urine; Plasma;

Bacopaside I is one of the main pseudojujubogenin glycosides isolated from Bacopa monniera. In the present study, a rapid and robust LC-ESI–MS/MS method was developed and validated to quantify bacopaside I in rat plasma. After plasma samples were deproteinized by methanol, the post-treatment samples were analyzed on a Zorbax Eclipse Plus C18 (2.1 × 50 mm, 1.8 μm) column using a mobile phase of acetonitrile and water (65:35, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer with selected reaction monitoring (SRM) mode via electrospray ionization source. This method covered a linearity range of 10–2000 ng/mL with the lower limit of quantification of 10 ng/mL. The intra- and inter-day precisions of analysis were less than 10.2%, and the accuracies were between −11.1% and 8.4% at the concentrations of 25, 150 and 1800 ng/mL. The total run time was 6.0 min. This method was successfully applied to the preclinical pharmacokinetic study of bacopaside I following intravenous or oral administration to rats.
Keywords: Bacopaside I; LC-ESI–MS/MS; Rat plasma; Pharmacokinetics;

Development of a modified QuEChERS method for the determination of veterinary antibiotics in swine manure by liquid chromatography tandem mass spectrometry by Chunna Guo; Mingru Wang; Hui Xiao; Binbin Huai; Feng Wang; Guangfang Pan; Xiaoping Liao; Yahong Liu (110-118).
A QuEChERS (quick, easy, cheap, effective, rugged and safe) based methodology was developed for the rapid, simultaneous quantification and identification of 26 veterinary drugs in swine manure by liquid chromatography tandem mass spectrometry. The selected antibiotics included tetracyclines, sulfonamides, macrolides, fluoroquinolones, lincosamides and pleuromutilins. This is the first study to determine pleuromutilin levels in manure. The QuEChERS process involved two simple steps. First, sample extraction with methanol: acetonitrile: 0.1 M EDTA-McIlvaine buffer followed by phase separation with MgSO4: NaCl addition. The supernatant was then extracted and cleaned by dispersive solid-phase extraction using a primary-secondary amine (PSA) and octadecylsilane (C18) support. The proposed method provides a linearity in the range of 1–500 ng mL-1 and linear regression coefficients (r) were greater than 0.996. MDL and MQL ranged between 0.01–1.86 μg kg−1 and 0.05–5.91 μg kg−1, respectively. Recoveries ranged from 61.39 to 105.65% with the exception of sulfaquinoxaline (55.7–56.8%) and valnemulin (33.7–37.7%). This method resulted in good precision (repeatability and reproducibility) and relative standard deviations less than 17% within the same day, and lower than 20% between days. The method was then applied to study the swine manure samples collected from Guangdong, China. Chlortetracycline, tetracycline, doxycycline, sulfadimidine and tilmicosin were detected in all samples indicating high residuals in manure. In fact tilmicosin was detected at 14400 μg kg−1 suggesting that prudent treatment of manure should be conducted to prevent environmental contamination. In conclusion, this workflow can provide a simpler and more cost-effective alternative to conventional methods and is compatible with processing large sample numbers over a short time period.
Keywords: QuEChERS; Veterinary antibiotics; Liquid chromatography tandem mass; Swine manure;

Gushudan, a Chinese compound formulation based on the theory of traditional Chinese medicine and desgined to treat osteoporosis. However, its intergated intervention effective mechanism in vivo is not well understood. In this study, an intergated serum and urinary metabonomic strategy based on UPLC-MS technique have been developed to increase the understanding of the metabolism characters of osteoporosis and to investigate the holistic therapeutic efficacy of Gushudan on prednisolone-induced osteoporosis rat model. Principle component analysis (PCA) was utilized to identify differences in metabolic profiles of rats in the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group and clear separation was achieved among three groups. Furthermore, 17 potential biomarkers from urine and 10 potential biomarkers from serum were identified, primiarily related to amino acid metabolism, energy metabolism, lipid metabolism, intestinal flora metabolism and kidney damage. Gushudan has therapeutic effects on rat with osteoporosis via the regulation of multiple metabolic pathways. It’s worth mentioning that some new biomarkers associated with osteoporosis such as 3-methoxydopa, 2,8-digydroxyadenine have been discovered in this study for the first time. This study provides a useful approach to get insight into the intergated metabonomic mechanism of prednisolone-induced osteoporosis and to assess the efficacy of Gushudan on osteoporotic rats. The work also shows that the metabonomics method is a promising tool in the efficacy and mechanism research of traditional Chinese compound medicines.
Keywords: Gushudan; Osteoporosis; UPLC-MS; Metabonomics; Principle component analysis;

Whether as a rodenticide or as a natural product, monofluoroacetic acid (FAcOH) may cause poisoning to humans or animals for its high acute toxicity. Urine is one of the most typical specimens for forensic diagnosis when poisoning case about FAcOH happens. The positive sample containing FAcOH plays a key role for the development of an accurate and reliable analytical method. The bioconversion from monofluoroacetamide (FAcNH2) to FAcOH in urine in vitro was studied for the preparation of positive urine sample containing FAcOH without standard spiking or animal experiment. The average bioconversion rates were 0%, 18.6% and 41.3% when incubated the FAcNH2 spiked urine in vitro for 21 days at −20 °C, room temperature (RT) and 37 °C, respectively. Afterwards, a fast and sensitive analytical method was developed for determination of FAcOH in urine. Samples were diluted with water containing formic acid and cleaned with polymeric anion exchange (PAX) cartridge. The acid eluate was neutralized with ammonium hydroxide and directly measured by hydrophilic interaction liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) using basic mobile phase condition. The limit of detection and limit of quantification of FAcOH in urine were 2 and 5 ng mL−1, respectively. The linear range was 5–1000 ng mL−1 with a correlation coefficient of r = 0.9993 in urine calibrated with internal standard. The recoveries at four spiking levels (5, 10, 50 and 500 ng mL−1 in urine) were 87.2%–107% with relative standard deviations ranged between 4.3%–8.8%.
Keywords: Monofluoroacetic acid; Monofluoroacetamide; Bioconversion in vitro; Urine; Liquid chromatography–triple quadrupole mass spectrometry;

In this study, it was the first time that we simultaneously quantified nimesulide and its possible metabolites M1, M2, M3 and M4 by employing liquid chromatography-tandem mass spectrometry (LC–MS/MS). Nimesulide-d5 was used as internal standard (IS) for validation. Analytes and IS were recovered from human plasma by protein precipitation with acetonitrile. Prepared plasma samples were analyzed under the same LC–MS/MS conditions, and chromatographic separation was realized by using an Ultimate C18 column, with run time being 5 min for each sample. Our results showed that various analytes within their concentration ranges could be quantified accurately by using the method. Mean intra- and inter-day accuracies ranged from −4.8% to 4.8% (RE), and intra- and inter-assay precision ≤6.2% (RSD). The following parameters were validated: specificity, recovery, matrix effects, dilution integrity, carry-over, sample stability under a variety of storage and handling conditions (room temperature, freezer, freeze-thaw and post-preparative) and stock solution stability. Pharmacokinetics of nimesulide and its metabolites were calculated based on the analysis of samples collected from twelve Chinese healthy volunteers after single oral dose of 100 mg nimesulide tablets. By applying the pharmacokinetic determination into human samples, we preliminarily detected a new metabolite of nimesulide (M4*), and the concentration of M4* was relatively higher in plasma. Furthermore, we predicted part of conceivable metabolism pathway in plasma of after oral administration of 100 mg nimesulide tablets. This research provided an experimental basis for further studies on metabolic activation and biotransformation of nimesulide, and for more comprehensive conjecture of its metabolic pathways.
Keywords: Nimesulide; Metabolites; LC–MS/MS; Pharmacokinetics;

Selective enrichment and desalting of hydrophilic peptides using graphene oxide by Miao Jiang; Linyu Qi; Peiru Liu; Zijun Wang; Zhigui Duan; Ying Wang; Zhonghua Liu; Ping Chen (149-157).
The wide variety and low abundance of peptides in tissue brought great difficulties to the separation and identification of peptides, which is not in favor of the development of peptidomics. RP-HPLC, which could purify small molecules based on their hydrophobicity, has been widely used in the separation and enrichment of peptide due to its fast, good reproducibility and high resolution. However, RP-HPLC requires the instrument and expensive C18 column and its sample capacity is also limited. Recently, graphene oxide has been applied to the adsorption of amino acids. However, the enrichment efficiency and selectivity of graphene oxide for peptides remain unclear. In this study, the adsorption efficiency and selectivity of graphene oxide and RP-C18 matrix were compared on trypsinized α-actin and also on tissue extracts from pituitary gland and hippocampus. For α-actin, there exhibit similar elution peaks for total trypsinized products and those adsorpted by GO and C18 matrix. But peptides adsorbed by GO showed the higher hydrophilic peaks than which adsorbed by C18 matrix. The resulted RP-HPLC profile showed that most of peptides enriched by graphene oxide were eluted at low concentration of organic solvent, while peptides adsorbed by RP-C18 matrix were mostly eluted at relatively high concentration. Moreover, mass spectrometry analysis suggested that, in pituitary sample, there were 495 peptides enriched by graphene oxide, 447 peptides enriched by RP-C18 matrix while in hippocampus sample 333 and 243 peptides respectively. The GRAVY value analysis suggested that the graphene oxide has a stronger adsorption for highly hydrophilic peptides compared to the RP-C18 matrix. Furthermore, the combination of these two methods could notably increase the number of identification peptides but also the number of predicted protein precursors. Our study provided a new thought to the role of graphene oxide during the enrichment of peptides from tissue which should be useful for peptidomics study.
Keywords: Graphene oxide; Selective enrichment; Hydrophilic peptide; Solid-phase extraction;

A liquid chromatography–tandem mass spectrometry (LC–MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1 mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150 × 3.9 mm, 5 μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5 min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449–252 for tolvaptan, m/z 479–252 for metabolite DM-4103, m/z 481–252 for metabolite DM-4107 and m/z 463–266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1 ng/mL. The method was validated over a linear range from 1 to 500 ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15 mg tolvaptan tablet.
Keywords: Tolvaptan; Metabolite; LC–MS/MS; Human plasma; Method validation;

In this study a novel method is described for selective quantization of domperidone in biological matrices applying molecular imprinted polymers (MIPs) as a sample clean up procedure using high performance liquid chromatography coupled with a fluorescence detector. MIPs were synthesized with chloroform as the porogen, ethylene glycol dimethacrylate as the crosslinker, methacrylic acid as the monomer, and domperidone as the template molecule. The new imprinted polymer was used as a molecular sorbent for separation of domperidone from serum. Molecular recognition properties, binding capacity and selectivity of MIPs were determined. The results demonstrated exceptional affinity for domperidone in biological fluids. The domperidone analytical method using MIPs was verified according to validation parameters, such as selectivity, linearity (5–80 ng/mL, r2  = 0.9977), precision and accuracy (10–40 ng/mL, intra-day = 1.7–5.1%, inter-day = 4.5–5.9%, and accuracy 89.07–98.9%).The limit of detection (LOD) and quantization (LOQ) of domperidone was 0.0279 and 0.092 ng/mL, respectively. The simplicity and suitable validation parameters makes this a highly valuable selective bioequivalence method for domperidone analysis in human serum.
Keywords: HPLC; Human serum; Domperidone; Molecular imprinted polymer; Selective extraction;

The development and application of a novel LC–MS/MS method for the measurement of Dolutegravir, Elvitegravir and Cobicistat in human plasma by Sujan Dilly Penchala; Sandra Fawcett; Laura Else; Deirdre Egan; Alieu Amara; Emilie Elliot; Elizabeth Challenger; David Back; Marta Boffito; Saye Khoo (174-180).
Dolutegravir and Elvitegravir belongs to a class of integrase inhibitors which has recently been approved by the FDA for the treatment of HIV-infection. Elvitegravir and its co-administered booster drug, Cobicistat, has shown the potential to be a candidate for a one pill once a day regimen and is currently a component of many clinical trials. A sensitive LC–MS/MS method has been developed and validated for the simultaneous determination of these three drugs in human plasma. A liquid- liquid extraction was used as a sample preparation technique using 100 μL of plasma. The method was validated from 10 to 4000 ng/mL for Dolutegravir, Elvitegravir and Cobicistat. Chromatography was performed on XBridge C18 2.1 mm × 50 mm column, using an 80:20 methanol/water mobile phase containing 0.1% formic acid on a gradient program. This method was successfully applied for ongoing clinical trials.
Keywords: Dolutegravir; Elvitegravir; Cobicistat; LC–MS/MS; Method validation;

Physiologically relevant plasma d,l-homocysteine concentrations mobilize Cd from human serum albumin by Peter Sagmeister; Matthew A. Gibson; Kyle H. McDade; Jürgen Gailer (181-186).
Although low-level chronic exposure of humans to cadmium (Cd2+) can result in a variety of adverse health effects, little is known about the role that its interactions with plasma proteins and small molecular weight (SMW) ligands in the bloodstream may play in delivering this metal to its target organs. To gain insight, a Cd-human serum albumin (HSA) 1:1 (molar ratio) complex was analyzed by size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using a phosphate buffered saline (PBS)-buffer mobile phase, the stability of the Cd-HSA complex was investigated in the presence of 2.0 mM of SMW ligands, including taurine, acetaminophen, l-methionine, l-cysteine (Cys), d,l-homocysteine (hCys) or l-cysteine methyl-ester (Cys-Me). While taurine, acetaminophen and l-methionine did not affect its integrity, Cys, hCys and Cys-Me completely abstracted Cd from HSA. Subsequent investigations into the effect of 1.5, 1.0 and 0.5 mM Cys and hCys on the integrity of the Cd-HSA complex revealed clear differences with regard to the nature of the eluting SMW-Cd species between these structurally related endogenous thiols. Interestingly, the Cd-specific chromatograms that were obtained for 0.5 mM hCys revealed the elution of an apparent mixture of the parent Cd-HSA complex with a significant contribution of a structurally uncharacterized CdxhCysy species. Since this hCys concentration is encountered in blood plasma of hyperhomocysteinemia patients and since previous studies by others have revealed that a SH-containing carrier mediates the uptake of Cd into hepatocytes, our results suggest that plasma hCys may play a role in the toxicologically relevant translocation of Cd from the bloodstream to mammalian target organs.
Keywords: Albumin; Cadmium; Cysteine; Homocysteine; SEC; Hyphenation; Cd-specific detection; Hyperhomocysteinemia;

A simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was firstly developed and validated for simultaneous determination of netupitant and palonosetron in human plasma using ibrutinib as the internal standard (IS). Following liquid–liquid extraction, the compounds were eluted isocratically on a Phenomenex C18 column (50mm × 2.0 mm, 3 μm) with the mobile phase consisting of acetonitrile and 10 mM ammonium acetate buffer (pH 9.0) (89:11, v/v) at the flow rate of 0.3 mL/min. The monitored ion transitions were m/z 579.5 → 522.4 for netupitant, m/z 297.3 → 110.2 for palonosetron and m/z 441.2 → 138.1 for IS. Chromatographic run time was 2.5 min per injection, which made it possible to analyze more than 300 of samples per day. The assay exhibited a linear dynamic range of 5–1000 ng/mL for netupitant and 0.02–10 ng/mL for palonosetron in plasma. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). Selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect, recovery and carry-over effect were evaluated for all analytes. The method is simple, rapid, and has been applied successfully to a pharmacokinetic study of netupitant and palonosetron in healthy volunteers.
Keywords: Netupitant; Palonosetron; LC–MS/MS; Human plasma; Pharmacokinetics;

High gradient magnetic field microstructures for magnetophoretic cell separation by Abdel Rahman Abdel Fattah; Suvojit Ghosh; Ishwar K. Puri (194-199).
Microfluidics has advanced magnetic blood fractionation by making integrated miniature devices possible. A ferromagnetic microstructure array that is integrated with a microfluidic channel rearranges an applied magnetic field to create a high gradient magnetic field (HGMF). By leveraging the differential magnetic susceptibilities of cell types contained in a host medium, such as paramagnetic red blood cells (RBCs) and diamagnetic white blood cells (WBCs), the resulting HGMF can be used to continuously separate them without attaching additional labels, such as magnetic beads, to them. We describe the effect of these ferromagnetic microstructure geometries have on the blood separation efficacy by numerically simulating the influence of microstructure height and pitch on the HGMF characteristics and resulting RBC separation. Visualizations of RBC trajectories provide insight into how arrays can be optimized to best separate these cells from a host fluid. Periodic microstructures are shown to moderate the applied field due to magnetic interference between the adjacent teeth of an array. Since continuous microstructures do not similarly weaken the resultant HGMF, they facilitate significantly higher RBC separation. Nevertheless, periodic arrays are more appropriate for relatively deep microchannels since, unlike continuous microstructures, their separation effectiveness is independent of depth. The results are relevant to the design of microfluidic devices that leverage HGMFs to fractionate blood by separating RBCs and WBCs.
Keywords: Magnetophoresis; Label-free separation; Microstructures; Magnetism; Microfluidics;

3-Hydroxybenzo[a]pyrene (3-OHBaP) is widely used as a biomarker for assessing carcinogenic benzo[a]pyrene exposure risks. However, monitoring urinary 3-OHBaP suffers from an insufficient sensitivity due to the pg/mL level in urine excretion. In this study, a sensitive method for determination trace urinary 3-OHBaP was developed, involving enzymatic hydrolysis of the glucuronide and sulfate conjugates, ionic liquids dispersive liquid–liquid microextraction (IL-DLLME) enrichment, derivatization with dansyl chloride and HPLC-HRMS/MS analysis in the positive ion mode. Using IL-DLLME makes the enrichment of trace 3-OHBaP very simple, time-saving, efficiency and environmentally-friendly. To enhanced HPLC-HRMS/MS response, an MS-friendly dansyl group was introduced to increase the ionization and fragmentation efficiency. The optimal IL-DLLME extraction parameters and derivatization reaction conditions were investigated. Good linearity was obtained over a concentration range of 0.6–50.0 pg/mL with correlation coefficients (r2) of 0.9918. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.2 pg/mL and 0.58 pg/mL, respectively. The recoveries were 92.0 ± 4.2% with the intra-day and inter-day RSD values ranged from 2.2% to 3.8% and from 3.3% to 6.8%, respectively. The proposed IL-DLLME-Dansylation-HPLC-HRMS/MS method was successfully applied to determine urinary 3-OHBaP of non-occupational exposed smokers and nonsmokers.
Keywords: 3-Hydroxybenzo[a]pyrene; Ionic liquid dispersive liquid–liquid microextraction; Chemical derivatization; Human urine; HPLC-HRMS/MS;

Display OmittedChlorzoxazone is a probe drug to assess cytochrome P450 (CYP) 2E1 activity (phenotyping). If the pharmacokinetics of the probe drug is linear, pharmacologically ineffective doses are sufficient for the purpose of phenotyping and adverse effects can thus be avoided. For this reason, we developed and validated an assay for the ultrasensitive quantification of chlorzoxazone and 6-hydroxychlorzoxazone in human plasma. Plasma (0.5 mL) and liquid/liquid partitioning were used for sample preparation. Extraction recoveries ranged between 76 and 93% for both analytes. Extracts were separated within 3 min on a Waters BEH C18 Shield 1.7 μm UPLC column with a fast gradient consisting of aqueous formic acid and acetonitrile. Quantification was achieved using internal standards labeled with deuterium or 13C and tandem mass spectrometry in the multiple reaction monitoring mode using negative electrospray ionization, which yielded lower limits of quantification of 2.5 pg mL−1, while maintaining a precision always below 15%. The calibrated concentration ranges were linear for both analytes (2.5–1000 pg mL−1) with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <15% and <11% and plasma matrix effects always were below 50%. The assay was successfully applied to assess the pharmacokinetics of chlorzoxazone in two human volunteers after administration of single oral doses (2.5–5000 μg). This ultrasensitive assay allowed the determination of chlorzoxazone pharmacokinetics for 8 h after microdosing of 25 μg chlorzoxazone.
Keywords: Chlorzoxazone; 6-hydroxychlorzoxazone; CYP2E1; Microdosing; Ultra high performance liquid chromatography; Tandem mass spectrometry;

In this investigation, a novel strategy for separation and quantitative determination of four metabolites of cinacalcet (M2a-Glu, M2b-Glu, M7-Gly, and M8-Gly) in human urine is suggested. The analytical assay is based on a pre-column derivation procedure of cinacalcet metabolites with 1-pyrenyldiazomethane (PDAM) as a fluorescent labeling reagent, and subsequently separation and quantitative determination with reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. Metabolites were separated on a Microsorb-MV 100-5 C18 chromatography column (250 × 4.6 mm, 5 μm) using acetate buffer (pH 3.5):methanol (30:70 v/v) as mobile phase at a flow rate of 1.0 mL min−1. The method was fully validated in terms of linearity (r2  > 0.996; 1–10 ng mL−1), precision (both intra-day and inter-day; RSD < 6.2%), accuracy (92–110%), specificity, robustness (0.15% < RSD < 4.1%), limits of detection (5 × 10−4 to 3 × 10−3  ng mL−1) and quantification (2 × 10−3 to 1 × 10−2  ng mL−1). According to the results, the proposed method can be useful in the routine analysis for the determination of cinacalcet metabolites in urine samples.
Keywords: Cinacalcet; Metabolites; Fluorescent labeling; Urine; RP-HPLC; Validation;

A sensitive and convenient high performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method was developed to determine levophencynonate and demethyl levophencynonate levels in human plasma simultaneously. Chromatographic separation was achieved on a SHIMADZU Shim-Pack XR C8 column and mass spectrometric analysis was performed by an API5000 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 358.4 → 156.4 and 344.5 → 144.2 were used to quantify levophencynonate and demethyl levophencynonate, respectively. This analytical method was fully validated with specificity, linearity, lower limit of quantitation (LLOQ), accuracy, precision, stability, matrix effect and recovery. The linearity of this method were developed to be within the concentration ranges of 10–4000 pg/mL for levophencynonate and 25–8000 pg/mL for demethyl levophencynonate in human plasma. This method was used in a clinical study which was administrated with single oral dose for Chinese healthy subjects to investigate the pharmacokinetics of levophencynonate and demethyl levophencynonate.
Keywords: Levophencynonate; Demethyl levophencynonate; HPLC–MS/MS; Human plasma;

Tofogliflozin is a novel selective inhibitor of sodium-dependent glucose co-transporter-2 (SGLT2) and has been developed for the treatment of patients with type 2 diabetes mellitus. In this study, a highly sensitive and specific liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantitation of tofogliflozin in rat plasma was developed and validated. The detection was performed using an API 3200 triple-quadrupole mass spectrometer with selected reaction monitoring (SRM) in the positive electrospray ionization mode. The SRM transitions were m/z  = 387.1 [M+H]+  → 267.1 for tofogliflozin and m/z  = 451.2 [M+H]+  → 71.0 for empagliflozin (internal standard: I.S.). Chromatographic separation was performed on a Quicksorb ODS (2.1 mm i.d. × 150 mm, 5 μm size) using isocratic elution with acetonitrile/10 mM ammonium acetate (50:50, v/v) as the mobile phase at a flow rate of 0.2 mL/min and the total run time was 4.0 min. The lower limit of quantification (LLOQ) for tofogliflozin was 0.5 ng/mL with sufficient specificity, accuracy, and precision. The validated method was successfully applied to the pharmacokinetic studies of tofogliflozin in rats. This assay method could be a valuable tool for future studies including pharmacokinetic and pharmacodynamic studies of SGLT2 inhibitors.
Keywords: LC–MS/MS; Tofogliflozin; Rat plasma; Antidiabetic agent;

Development and validation of a UPLC–MS/MS method for quantitation of droxidopa in human plasma: Application to a pharmacokinetic study by Haidong Wang; Guangsheng Yang; Jinyu Zhou; Jiang Pei; Qiangfeng Zhang; Xingfa Song; Zengxian Sun (234-238).
In this study, a simple and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for quantitation of droxidopa in human plasma for the first time. A simple plasma protein precipitation method using methanol containing 3% formic acid was selected, and the separation was achieved by an Acquity UPLC™ BEH Amide column (2.1 mm × 50 mm, 1.7 μm) with a gradient elution using acetonitrile, ammonium formate buffer and formic acid as mobile phase. The detection of droxidopa and benserazide (internal standard, IS) was performed using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The precursor-to-product ion transitions m/z 214.2 →  m/z 152.0 for droxidopa, and m/z 258.1 →  m/z 139.1 for IS were used for quantification. A lower limit of quantification of 5.00 ng/mL was achieved and the linear curve range was 5.00–4000 ng/mL using a weighted (1/x2) linear regression model. Intra-assay and inter-assay precision was less than 10.2%, and the accuracy ranged from 0.1% to 2.1%. Stability, recovery and matrix effects were within the acceptance criteria recommended by the regulatory bioanalytical guidelines. The method was successfully applied to a pharmacokinetic study of droxidopa in healthy Chinese volunteers.
Keywords: Droxidopa; UPLC–MS/MS; Plasma concentrations;

A stereoselective assay was developed for the quantification of bupropion and oxidative, reductive, and glucuronide metabolites (16 analytes total) in human urine. Initially, authentic glucuronide standards obtained from commercial sources were found to be incorrectly labeled with regard to stereochemistry; the correct stereochemistry was unequivocally reassigned. A trifurcated urine sample preparation and analysis procedure was employed for the stereoselective analysis of bupropion, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion enantiomers, and hydroxybupropion, erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers in urine. Method 1 stereoselectively analyzed bupropion (R and S), and unconjugated free hydroxybupropion (R,R and S,S), erythrohydrobupropion (1R,2S and 1S,2R), and threohydrobupropion (1R,2R and 1S,2S) using chiral chromatography with an α1-acid glycoprotein column. Because no hydroxybupropion β-d-glucuronide standards were commercially available, method 2 stereoselectively analyzed total hydroxybupropion aglycones (R,R and S,S-hydroxybupropion) after urine hydrolysis by β-glucuronidase. Hydroxybupropion β-d-glucuronide (R,R and S,S) urine concentrations were calculated as the difference between total and free hydroxybupropion (R,R and S,S) concentrations. Due to incomplete β-glucuronidase hydrolysis of erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers, method 3 stereoselectively analyzed intact erythrohydrobupropion and threohydrobupropion β-d-glucuronide diastereomers using C18 column chromatography. All analytes were quantified by positive ion electrospray tandem mass spectrometry. The assay was fully validated over analyte-specific concentrations. Intra- and inter assay precision were within 15% for each analyte. The limits of quantification for bupropion (R and S), hydroxybupropion (R,R and S,S), threohydrobupropion (1S,2S and 1R,2R), erythrohydrobupropion (1R,2S and 1S,2R) were 10, 50, 100, and 100 ng/mL, respectively. The limits of quantification for (1R,2R)-threohydrobupropion β-d-glucuronide, (1S,2S)-threohydrobupropion β-d-glucuronide, and (1R,2R)-erythrohydrobupropion β-d-glucuronide were each 50 ng/mL. Due to the abundance of bupropion and metabolites in human urine, no efforts were made to optimize sensitivity. All analytes were stable following freeze thaw cycles at −80 °C. This assay was applicable to clinical pharmacokinetic investigations of bupropion in patients and to in vitro metabolism of the primary bupropion metabolites to their glucuronides.
Keywords: Bupropion; Urine; LC–MS/MS; Analytical method; Stereoselectivity;