Journal of Chromatography B (v.1004, #C)

The concept of personalized medicine is related to the development of new sensitive, precise and accurate analytical methods for therapeutic drug monitoring. In this article a rapid, sensitive and specific method was developed for the quantification of aliskiren, losartan, valsartan and hydrochlorothiazide in human plasma. Sample preparation was performed by protein precipitation with acetonitrile followed by filtration. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 μm) under isocratic elution. The mobile phase was composed of a 10% 5 mM ammonium formate water solution pH 4.5, adjusted with formic acid, in acetonitrile and pumped at a flow rate of 0.25 mL min−1. The assay was linear over the concentration range of 5–500 ng mL−1 for all the analytes. Intermediate precision was less than 5.2% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis antihypertensives in human plasma. With a small sample size (50 μL human plasma) and a run time less than 6.0 min for each sample the method can be used to support a wide range of clinical studies and therapeutic drug monitoring.
Keywords: Direct injection analysis; Hydrophilic interaction liquid chromatography; Mass spectrometry; Therapeutic drug monitoring; Antihypertensive drugs; Human plasma;

One-step separation of nine structural analogues from Poria cocos (Schw.) Wolf. via tandem high-speed counter-current chromatography by Hualiang Zeng; Qi Liu; Jingang Yu; Xinyu Jiang; Zhiliang Wu; Meiling Wang; Miao Chen; Xiaoqing Chen (10-16).
A novel one-step separation strategy—tandem high-speed counter-current chromatography (HSCCC) was developed with a six-port valve serving as the switch interface. Nine structural analogues including three isomers were successfully isolated from Poria cocos (Schw.) Wolf. by one step. Compared with conventional HSCCC, peak resolution of target compounds was effectively improved in tandem one. Purities of isolated compounds were all over 90% as determined by HPLC. Their structures were then identified via UV, MS and 1H NMR, and eventually assigned as poricoic acid B (1), poricoic acid A (2), 3β,16α-dihydroxylanosta-7, 9(11), 24-trien-21-oic acid (3), dehydrotumulosic acid (4), polyporenic acid C (5), 3-epi-dehydrotumulosic acid (6), 3-o-acetyl-16α-hydroxydehydrotrametenolic acid (7), dehydropachymic acid (8) and dehydrotrametenolic acid (9) respectively. The results indicated that tandem HSCCC can effectively improve peak resolution of target compounds, and can be a good candidate for HSCCC separation of structural analogues.
Keywords: Tandem; HSCCC; Structural analogues; Extrusion elution; Poria cocos (Schw.) Wolf.;

Simultaneous qualitation and quantitation of natural trans-1,4-polyisoprene from Eucommia ulmoides Oliver by gel permeation chromatography (GPC) by Tianyang Guo; Yuanbo Liu; Yanxiu Wei; Xiang Ma; Qiuming Fan; Jun Ni; Zequn Yin; Jiahui Liu; Sai Wang; Yiyang Dong; Jichuan Zhang; Liqun Zhang; Haijia Su; Tianwei Tan (17-22).
Natural trans-1,4-polyisoprene (TPI) as a functional biomaterial has aroused great interest for rubber industrial product use. Here, we proposed a method that enables simultaneous analysis of the content and molecular-weight distribution (MWD) of natural TPI by gel permeation chromatography (GPC). The natural TPIs were collected from leaves, fruit coatings and bark of Eucommia ulmoides Oliver (E. ulmoides) through toluene extraction followed by ethanol purification. The results of TPI contents from leaves and fruit coatings were shown ca. 3.5% and 13.8%, respectively. Accordingly, limits of detection (LODs) of TPI were 0.58 mg/mL from leaves and 0.47 mg/mL from fruit coatings. The MWDs of TPI demonstrated a bimodal distribution from leaves, a unimodal distribution from bark, and a unimodal distribution with a tiny peak shoulder from fruit coatings. In real-life E. ulmoides analysis, the results from three independent methods (GPC, gravimetric method, and infrared spectroscopy) were obtained with good consistency.
Keywords: trans-Polyisoprene (TPI); Quantitation; Molecular-weight distribution (MWD); Eucommia ulmoides oliver; Gel permeation chromatography (GPC);

In the current study, a novel method for high-throughput and sensitive determination of 12 phytohormones in plants was developed by using sequential solvent induced phase transition extraction (SIPTE) coupled with ultra–performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). In sequential SIPTE, 0.1% formic acid (v/v) and 50 mM NaHCO3 aqueous solution were used for enrichment and purification of alkaline and acidic phytohormones from the acetonitrile extract of plant tissues in sequence, in which hydrophobic solvent (toluene) was added to the acetonitrile aqueous mixture for driving the phase separation. Under optimized sequential SIPTE conditions, the phytohormones in acetonitrile extract of plant tissues could be effectively enriched and purified, which was in favor of the following UPLC–MS/MS analysis with less matrix effect. The phytohormones could be detected using the developed sequential SIPTE–UPLC–MS/MS method with the limits of the detection (LODs) ranging from 0.56 to 438.60 pg mL−1 and linear range over 2 orders of magnitude with correlation coefficients (r) > 0.9970. The relative recoveries of the detected phytohormones were in the range of 85.1–114.6%. Finally, the proposed method was applied to simultaneous determination of endogenous phytohormones in different tissues of model plants (Oryza sativa and Arabidopsis thaliana) with small amount of sample size (5 mg, fresh weight). The proposed method may be suitable for studying the distribution of phytohormones in model plants.
Keywords: Phytohormones; Sample pretreatment; SIPTE; UPLC–MS/MS;

Capillary electrophoresis coupled with 1,1′-thiocarbonyldiimidazole derivatization for the rapid detection of total homocysteine and cysteine in human plasma by Alexander Vladimirovich Ivanov; Edward Danielevich Virus; Boris Petrovich Luzyanin; Aslan Amirkhanovich Kubatiev (30-36).
A simple and rapid approach is described for the determination of total plasma cysteine and homocysteine using capillary electrophoresis. Human plasma samples were reduced with dithiothreitol and then processed with 1,1′-thiocarbonyldiimidazole in acetonitrile. After centrifugation, the sample supernatant was injected directly into a capillary by applying negative voltage and analytes were stacked after alkaline post-injection. Using a 50 μm i.d. silica capillary of 35 cm total length, filled with 0.1 M triethanolamine, 0.15 M formic acid, and 50 μM hexadecyltrimethylammonium bromide (pH 3.9), we reached a limit of quantification of 2.5 μM for homocysteine. Accuracy was 94.7–105.1%, intra- and inter-day imprecisions were <2.5 and <3%, respectively. The total analysis time was 6 min. Furthermore, liquid-phase extraction with isopropanol led to a fourfold increase in sensitivity.
Keywords: Capillary electrophoresis; Homocysteine; Cysteine; 1,1′-Thiocarbonyldiimidazole;

Penehyclidine hydrochloride (PHC) is an anticholinergic drug with both antimuscarinic and antinicotinic activity. In order to compare the pharmacokinetics of two administration routes (intravenous injection (i.v.) and intramuscular injection (i.m.)) of PHC, an improved High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) bioanalytical method was developed for the quantification of PHC in plasma and urine using verapamil as the internal standard (I.S.). Chromatography was performed using a Thermo Hypersil GOLD column (30 mm × 2.1 mm, 3 μm), with a gradient elution of 1‰ formic acid–10 mmol/L ammonium acetate and acetonitrile at 0.3 mL/min. Detection and quantitation were performed by electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M + H]+ MRM transition of PHC at m/z 316.2 → 128.3 was used for PHC quantitation, and the transition at m/z 454.6 → 303.2 was used to monitor I.S. The lower limit of quantification (LLOQ) was 0.05 ng/mL. The intraday precision was <6.71% and the interday precision was <11.69%. The pharmacokinetic parameters of i.v. and i.m. administration routes were as follows (i.v. vs i.m.): t 1/2 15.73 vs 17.24 h, T max 0.06 vs 0.26 h, AUC0–t 69.35 vs 67.90 h ng/mL, AUC0–inf 78.24 vs 79.67 h ng/mL, C max 37.5 vs 9.1 ng/mL, Ae0–24 h 22.7 vs 25.21 μg. There were no significant differences between parameters t 1/2 and AUC (P  > 0.05), but significant differences were observed in C max, T max and Ae0–24h between the two administration routes (P  < 0.05). The mean absolute bioavailability of the i.m. administration route was 98.4% (95% confidence interval, 93.4–103.6%). Safety results showed that PHC appeared to be well tolerated in both i.v. and i.m. administration routes and pharmacokinetic results showed that PHC was nearly completely absorbed via i.m. administration route.
Keywords: Penehyclidine hydrochloride (PHC); Comparative pharmacokinetics; Bioavailability; Intramuscular injection (i.m.); Intravenous injection (i.v.);

Display OmittedAnnonacin is an environmental neurotoxin identified in the pulp of several fruits of the Annonaceae family (for example in Annona muricata, Asimina triloba), whose consumption was linked with the occurrence of sporadic atypical Parkinsonism with dementia. Pharmacokinetic parameters of this molecule are unknown. A method for its quantification in Rat plasma was developed, using its analogue annonacinone as an internal standard. Extraction from plasma was performed using ethylacetate with a good recovery. Quantification was performed by UPLC–MS/MS in SRM mode, based on the loss of the γ-methyl-γ-lactone (−112 amu) from the sodium-cationized species [M + Na]+ of both annonacin and internal standard. The limit of quantification was 0.25 ng/mL. Despite strong matrix effects, a good linearity was obtained over two distinct ranges 0.25–10 ng/mL and 10–100 ng/mL. The intra- and inter-day precisions (RSD) were lower than 10%, while accuracy was within ±10%. This method was applied to a pharmacokinetic study in the Rat. After oral administration of 10 mg/kg annonacin, a C max of 7.9 ± 1.5 ng/mL was reached at T max 0.25 h; T 1/2 was 4.8±0.7 h and apparent distribution volume was 387.9 ± 64.6 L. The bioavailability of annonacin was estimated to be 3.2 ± 0.3% of the ingested dose.
Keywords: Acetogenin; Annonaceae; Annonacin; Bioavailability; Rat plasma; UPLC–MS/MS;

An elevated uric acid (UA) in urine or serum can affect renal function and blood pressure, which is an indicator of gout, cardiovascular and renal diseases, hypertension, etc. In this work, a new type of mixed matrix membrane (MMM), based on graphitic carbon nitrides (g-CNs) and hollow fiber (HF), was prepared and combined with solid phase microextraction (SPME) mode to determine UA in urine and serum followed by gas chromatography-mass spectrometry (GC/MS). The porous g-CNs were dispersed in ammonia, and then the exfoliated g-CNs nanosheets were held in the pores of HF by capillary forces and sonification. The prepared g-CNs modified HF (g-CNs-HF) was immersed in biofluid directly to extract UA with SPME mode and the solvent-free mode is convenient for further derivatization and analysis. To achieve the highest extraction efficiency (EF), main extraction and derivatization parameters, such as g-CNs-HF immobilizing time, sonification power and time of extraction, derivatization and desorption time, were optimized. Under the optimum extraction conditions, a favorable linearity of UA was obtained in the range 0.1–200 μg mL−1 with correlation coefficients higher than 0.9990, and the average recoveries at three spiked levels of UA in urine and serum ranged from 80.7% to 121.6%, from 84.7% to 101.1%, respectively. The obtained results demonstrated the developed g-CNs-HF-SPME is a simple, rapid, cost-effective, solvent-free method for the analysis of UA in biofluid.
Keywords: Uric acid (UA); Graphitic carbon nitrides (g-CNs); Hollow fiber (HF); Solid phase microextraction (SPME); Gas chromatography-mass spectrometry (GC/MS);

A fast and sensitive method for quantifying lumefantrine and desbutyl-lumefantrine using LC–MS/MS by A.V. Silva; N. Mwebaza; M. Ntale; L.L. Gustafsson; A. Pohanka (60-66).
A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for quantification of lumefantrine (LUM) and its metabolite desbutyl-lumefantrine (DBL) in human plasma. Sample preparation was done by protein precipitation using acetonitrile containing deuterated lumefantrine (LUM-d18) and desbutyl-lumefantrine (DBL-d9) as internal standards. Total chromatography time was 2.2 min using an Hypersil Gold C18 column (20 × 2.1 mm, 1.9 μm), with a gradient using 0.5% formic acid in water (mobile phase A) and 0.5% formic acid in methanol (mobile phase B) at a flow rate of 0.5 mL/min. The mass spectrometric quantification was performed in positive electro spray ionization (ESI+) mode using selected reaction monitoring (SRM). Measuring range was 21–529 ng/mL for LUM and 1.9–47 ng/mL for DBL in plasma. Inter- and intra-assay precision was within 10% coefficient of variation (CV) for all levels of both LUM and DBL. Accuracy was within ±10% for all levels of both LUM and DBL. This method requires 100 μL plasma volume and its short retention times allow a high throughput. Samples were stable for a week at +5 °C, and up to six months −20 °C. The method was successfully applied for plasma LUM and DBL determination in children under 5 years of age with uncomplicated malaria, up to 28 days after a standard 3-day treatment with artemether-lumefantrine.
Keywords: Desbutyl-lumefantrine; Liquid chromatography; Lumefantrine; Malaria; Plasma; Plasmodium falciparum; Sensivity; Tandem mass spectrometry;

Ketamine is a club drug widely abused for its hallucinogenic effects, being also used as a “date-rape” drug in recent years. We have developed an analytical method using gas chromatography-tandem mass spectrometry (GC–MS/MS) for the identification and quantification of ketamine and its major metabolite in urine and plasma. No derivatization step is needed to accomplish analysis. The compounds were extracted from 0.25 mL of sample using microextraction by packed sorbent on mixed mode (M1) cartridges. Calibration curves were linear in the range of 10–250 ng/mL for urine and 10–500 ng/mL for plasma, with determination coefficients higher than 0.99. The limit of detection (LOD) was 5 ng/mL for both compounds in both specimens. Recoveries ranged from 63 to 101%, while precision and accuracy were below 14% and 15%, respectively. These low limits of detection and the quite high recoveries obtained, in very low sample amounts, allow detecting small quantities of the compounds, making this procedure suitable for those laboratories performing routine analysis in the field of forensic toxicology. Compared with existing methods, the herein described procedure is fast, since no derivatization step is required, and cost effective for the quantification of ketamine and norketamine in biological specimens by gas chromatography.
Keywords: Ketamine; Norketamine; MEPS; Biological samples; GC–MS/MS;

A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025–2.0 μg/mL (R 2  > 0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025 μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50 mg rac-metoprolol.
Keywords: Metoprolol; Enantiomers; Chiral separation; Urine samples;

MS/MS and LC-MS/MS analysis of choline/ethanolamine plasmalogens via promotion of alkali metal adduct formation by Yurika Otoki; Kiyotaka Nakagawa; Shunji Kato; Teruo Miyazawa (85-92).
Tandem mass spectrometry (MS/MS) has been used for the analysis of plasmalogen (Pls), a physiologically important class of vinyl ether-linked phospholipid. However, MS/MS generally causes little fragmentation of Pls, especially choline Pls (PC-Pls). Previous MS/MS studies reported an increased formation of product ions of PC-Pls (and also ethanolamine Pls (PE-Pls)) in the presence of ‘alkali metals.’ Therefore, use of alkali metals considerably leads to the development of a method for analysis of both PC- and PE-Pls. In this study, this notion was evaluated using quadrupole-time-of-flight MS/MS and liquid chromatography (LC) coupled with MS/MS. Results from MS/MS confirmed that alkali metals (e.g., sodium) produced significant fragmentation of PC-Pls and PE-Pls. A number of structure-diagnostic product ions exhibiting high intensities were observed under optimized MS/MS conditions using alkali metals. Moreover, the ability to selectively and sensitively identify PC-Pls and PE-Pls at the molecular species level in biological samples (rat brain and heart) was demonstrated using LC-MS/MS. Therefore, the herein developed method appears to be a powerful tool for analyzing Pls and may provide a better understanding of their physiological roles in vivo.
Keywords: Plasmalogen; Choline plasmalogen; Ethanolamine plasmalogen; Tandem mass spectrometry; Rat tissues;

A simple method for the determination of Bosutinib in rat plasma by UPLC-MS/MS by Yihua Xu; Xin-ce Huang; Shengjie Dai; Yuwu Xiao; Meng-tao Zhou (93-97).
In this work, a simple, sensitive and fast ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitative determination of bosutinib in rat plasma. Plasma samples were processed with a protein precipitation. The separation was achieved by an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM). The validated method had an excellent linearity in the range of 0.1–500 ng/mL (R 2  > 0.9977) with a lower limit of quantification (0.1 ng/mL). The extraction recovery was in the range of 75.6–85.6% for bosutinib and 81.2% for pirfenidone (internal standard, IS). The intra- and inter-day precision was below 9.7% and accuracy was from −8.1% to 8.8%. No notable matrix effect and astaticism was observed for bosutinib. The method has been successfully applied to a pharmacokinetic study of bosutinib in rats for the first time, which provides the basis for the further development and application of bosutinib.
Keywords: Bosutinib; Rat; UPLC-MS/MS; Plasma; Pharmacokinetics;

Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide by Jianhua Zou; Xiaoqiong Ma; Guangji Zhang; Li Shen; Liting Zhou; Yu Yu; Fanfan Zhu; Zhe Chen (98-107).
Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI–MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile–water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1–5 μM iAsIII(AsIII ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAsIII is 1.0 μM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803.
Keywords: Sphingolipids; Arsenic trioxide; HPLC-ESI–MS/MS; Human multiple myeloma cell line U266; Human gastric cancer cell line MGC-803;