Journal of Chromatography B (v.877, #4)

A novel biological sample clean-up procedure has been developed for the determination of phosphorothioate oligodeoxynucleotides (PS-ODNs) and derived metabolites in biological fluids (plasma, urine and bile) and in tissues and feces from mice and rats. This method uses a one-step C18 solid-phase extraction (SPE) for biological matrix removal, and it uses capillary gel electrophoresis (CGE) for analyte detection. The assay is specific, and its linearity is superb (r  > 0.99) for IV-AS (a 13-mer PS-ODN) and PS19 (a 19-mer PS-ODN) in a variety of biological matrices. For both IV-AS and PS19, the precision, accuracy and absolute recovery values were found to be <20%, ±20% and 80–120%, respectively. The LODs of IV-AS and PS19 were 0.6 mg/l for plasma, 0.8 mg/l for rat urine and bile, 6 μg/g for rat tissues, and 10 μg/g for rat feces, with a signal-to-noise ratio of 3 (S/N  = 3). This method has been successfully applied to the analysis and quantitation of PS-ODNs in various biological samples arising from preclinical pharmacokinetic studies.
Keywords: Phosphorothioate oligodeoxynucleotides; Solid-phase extraction; CGE;

Development and validation of an LC–MS/MS method for the quantification of ephedrines in urine by K. Deventer; O.J. Pozo; P. Van Eenoo; F.T. Delbeke (369-374).
The objective of this study was to develop a simple and robust LC–MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d3, 4 μg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine–norephedrine and ephedrine–pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H2O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 μg/mL for norephedrine and norpseudoephedrine and 5 and 20 μg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from −5.5 to 12% for norephedrine, −4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC–NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.
Keywords: Doping; Urine; Ephedrine; Amphetamine; Sport; Mass spectrometry;

Organophosphorus flame retardants (OPFR), phosphorus triesters, are widely used chemicals with a high share of the worldwide flame retardant market. In animal experiments, dialkyl- and diarylphosphates are the main metabolites of OPFR. Therefore we elaborated a GC–MS/MS-method for the detection of OPFR-metabolites in human urine after solid phase extraction and derivatization with pentafluorobenzylbromide. The limits of detection range from 0.1 to 1 μg/l. Interday imprecision were 2–8%. The applicability of the method is shown by determination of the internal burden of 30 persons of the German general population. OPFR-metabolite concentrations range from <LOD to 27.5 μg/l for bis-(2-chlorethyl)-phosphate and <LOD to 4.1 μg/l for diphenylphosphate. Di-m-cresylphosphate and di-p-cresylphosphate cannot be detected in any of the native urine samples.
Keywords: Organophosphorus flame retardants; OPFR; Dialkyl phosphate metabolites; BCEP; DPP; DmCP; DpCP; TCEP; TPP; TCP;

An liquid chromatography–tandem mass spectrometry assay for determination of trace amount of new antifungal drug iodiconazole in human plasma by Shouhong Gao; Xia Tao; Lianna Sun; Chunquan Sheng; Wannian Zhang; Yunlei Yun; Jingxian Li; Haijun Miao; Wansheng Chen (382-386).
A simple, rapid and sensitive LC–MS/MS method for determination of trace amount of new antifungal drug iodiconazole in human plasma was developed (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[N-methyl-N-(3-chlor-benzyl)amino]-2-propanol), was used as internal standard (IS). The analytes were extracted by liquid–liquid extraction with n-hexane after internal standard spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with an isocratic mobile phase consisting of methanol–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The lower limit of quantification (LLOQ) was 0.10 ng/mL. This method was successfully used to determine the concentration of iodiconazole in human plasma following dermal administration.
Keywords: Iodiconazole; LC–MS/MS; Antifungal drug; Human plasma;

Plasma citrulline measurement using UPLC tandem mass-spectrometry to determine small intestinal enterocyte pathology by Pierre N.M. Demacker; Antonius M. Beijers; Henny van Daal; J. Peter Donnelly; Nicole M.A. Blijlevens; Johannes M.W. van den Ouweland (387-392).
Citrulline is a nonessential free amino acid, detectable in various biological fluids such as plasma, urine and cerebrospinal fluid. The plasma citrulline concentration is increasingly considered to be a reliable biomarker of enterocyte function. Current analysis usually involves lengthy HPLC separations as a part of classical amino acid profiling, or mass spectrometry usually in combination with derivatization. We employed UPLC-HILIC–tandem mass-spectrometry (MS/MS) of acetonitrile-derived supernatants from plasma samples of control subjects and of patients who had received myeloablative chemotherapy. Detection was achieved by the selected reaction monitoring of transitions: m/z 176 → 70 and 180 → 74 (for the deuterated standard), respectively. The method was precise and accurate with inter-day CV < 3.9% (n  = 30), recoveries ranging from 98.0 to 100.3% and high linearity from 0.3 to at least 2000 μmol/L. The results for 202 plasma samples agreed well with those obtained by the classical HPLC-fluorescence method. By a simple protein precipitation/extraction step and the UPLC separation the result can be available within 30 min of receipt with a capacity of at least 12 assays per hour. Citrulline in blood and plasma or serum was stable for at least 2 days at room temperature which would permit postal transport to the laboratory. The UPLC–MS/MS method for measuring plasma citrulline concentrations is fast and robust and is therefore an ideal tool for monitoring the intestinal enterocyte capacity of patients with various pathological conditions.
Keywords: Citrulline; UPLC; Mass spectrometry; Mucosal barrier injury; Amino acid marker; HILIC chromatography;

In the present study, a simple and efficient preconcentration method was developed using carrier mediated three phase liquid phase microextraction prior to HPLC–UV for simultaneous extraction and determination of trace amounts of highly hydrophilic tetracycline antibiotics including tetracycline (TCN), oxytetracycline (OTCN) and doxycycline (DCN) in bovine milk, human plasma and water samples. For extraction, 11.0 mL of the aqueous sample containing TCNs and 0.05 M Na2HPO4 (9.1 ≤ pH ≤ 9.5) was filled into a 12.0-mL vial. The solution of 1-octanol (containing 10% (w/v) of Aliquat-336 as carrier) was immobilized in the pores of a porous polypropylene hollow fiber. Aqueous receiving phase (RP, 24 μL of 0.1 M H3PO4 and 1.0 M NaCl with pH = 1.6) was located inside the lumen of hollow fiber and the fiber was transferred into the aqueous sample. After the extraction period, the receiving phase was directly injected into HPLC. In order to obtain high extraction efficiency, the parameters affecting the liquid phase microextraction were evaluated and optimized. Under the optimized conditions, the calibration curves were linear in the range of 0.5–1000 μg L−1 for TCN and OTCN, and in the range of 5–1000 μg L−1 for DCN with good linearity (r 2  > 0.995). Finally, applicability of the proposed method was successfully confirmed by extraction and determination of the drugs in water and plasma samples as well as in bovine milk samples with low and high fat contents. Comparing to the traditional methods, the proposed method exhibits high sensitivity and high preconcentration factors as well as good precision. The extraction setup is simple and due to active transport of analytes, high cleanup effect and good selectivity are obtained in extraction process.
Keywords: Tetracycline; Oxytetracycline; Doxycycline; Liquid phase microextraction; Carrier mediated extraction;

Bioassay guided discovery of apoptosis inducers from gamboge by high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry by Quan-Bin Han; Yan Zhou; Chao Feng; Gang Xu; Sheng-Xiong Huang; Song-Lin Li; Chun-Feng Qiao; Jing-Zheng Song; Donald C. Chang; Kathy Q. Luo; Hong-Xi Xu (401-407).
A screening system, composed of high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry, was established to find bioactive lead compound. This system succeeded in discovering apoptosis inducers from gamboge, the resin of Garcinia hanburyi. High-speed counter-current chromatography was used to provide well-separated fractions for bioassay and the resulted active fractions were rapidly identified using high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. The solvent system of n-hexane/ethyl acetate/methanol/water was optimized to the ratio of 7:3:7:3 (v/v/v/v) by a K value analysis. As a result, two active fractions were obtained. They showed apoptosis inducing effects as potent as that of taxol (500 nM) at the concentration of 1 μg/ml. Gambogenic acid (72.1%) and epimeric isogambogic acids (25.3%) were identified in one of the fractions. The other active fraction mainly contained two epimeric mixtures, gambogic acids (68.7%) and gambogoic acids (26.9%). Among them, gambogenic acid, without epimerization, has priority to be lead compound.
Keywords: High-speed counter-current chromatography; HPLC/MS; Apoptosis inducer; Garcinia hanburyi; Gambogenic acid;

Validation of a sensitive gas chromatographic–mass spectrometric method for the simultaneous determination of β-elemene and β-elemenal in human plasma by Zhihang Chen; Yanxia Song; Jinjing Che; Xiaolei Liu; Yu Ning; Chengqi Shan; Yunan Hou; Yunlong Liu; Xiaohong Miao; Yuanguo Cheng (408-414).
A sensitive gas chromatographic–mass spectrometric assay was described for determination of β-elemene and β-elemenal in human plasma, which has been successfully applied in clinical trial. After liquid–liquid extraction and gas chromatographic separation, the analytes were identified and quantitated. Calibration curves were linear in range from 31.25 to 8000 ng mL−1 and the limit of quantification for both was 31.25 ng mL−1. Intra- and inter-day precision at three concentrations were 2.3–8.3% with accuracy of −8.5 to 6.1% for elemene and 3.0–9.9% with accuracy of −2.3 to 5.9% for elemenal. The method was validated to be suitable for further pharmacokinetic study.
Keywords: β-Elemene; β-Elemenal; GC/MS; Pharmacokinetics;

Studies using in-tip solid phase microextraction (in-tip SPME) in a 96-well plate format are conducted to investigate the feasibility of SPME automation. The sample preparation process, including extraction and desorption, was fully automated and coupled with currently commercially available automated liquid handling systems. Several process parameters including extraction time and speed, and desorption time were investigated. An LC–MS/MS method has been developed and validated to determine the levels of a drug compound (MK-0533) in human plasma that demonstrates the suitability of this new approach. The developed method has a lower limit of quantitation (LLOQ) of 5 ng/mL when 0.25 mL of human plasma is processed and is validated in the concentration range of 5–2000 ng/mL. The successful application of the assay in clinical sample analysis indicates that in-tip SPME can be easily automated and has great potential to be used for high throughput quantitative determination of drugs in pharmaceutical industry.
Keywords: Automation; In-tip solid phase microextraction (in-tip SPME); 96-Well format; MK-0533; HPLC–MS/MS;

Determination of opiates and cocaine in urine by high pH mobile phase reversed phase UPLC–MS/MS by Thomas Berg; Elsa Lundanes; Asbjørg S. Christophersen; Dag Helge Strand (421-432).
A fast and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method for the determination of opiates (morphine, codeine, 6-monoacetylmorphine (6-MAM), pholcodine, oxycodone, ethylmorphine), cocaine and benzoylecgonine in urine has been developed and validated. Sample preparation was performed by solid phase extraction (SPE) on a mixed mode cation exchange (MCX) cartridge. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing of all analytes at the column inlet at gradient start, a basic mobile phase consisting of 5 mM ammonium bicarbonate, pH 10.2, and methanol (MeOH) was chosen. Positive electrospray ionization (ESI+) MS/MS detection was performed with a minimum of two multiple reaction monitoring (MRM) transitions for each analyte. Deuterium labelled-internal standards were used for six of the analytes. Between-assay retention time repeatabilities (n  = 10 series, 225 injections in total) had relative standard deviation (RSD) values within 0.1–0.6%. Limit of detection (LOD) and limit of quantification (LOQ) values were in the range 0.003–0.05 μM (0.001–0.02 μg/mL) and 0.01–0.16 μM (0.003–0.06 μg/mL), respectively. The RSD values of the between-assay repeatabilities of concentrations were ≤10% at five concentration levels for all analytes, except for pholcodine. Specificity was investigated by determination of the retention times of 96 drugs and internal standards in total. Co-eluting compounds were in all cases separated by the MS/MS detection. No or only minor matrix effects were observed. Total run time, including injection and equilibration time was 5.7 min. The method has been routinely used at the Norwegian Institute of Public Health (NIPH) since August 2007 for qualitative detection of opiates, cocaine and benzoylecgonine in more than 2000 urine samples with two replicates of each sample.
Keywords: UPLC; LC–MS/MS; Opiates; Opioids; Cocaine; High pH mobile phase; Reversed phase; Forensic toxicology;

Standardization of retention time data for AMT tag proteomics database generation by I.A. Tarasova; V. Guryča; M.L. Pridatchenko; A.V. Gorshkov; S. Kieffer-Jaquinod; V.V. Evreinov; C.D. Masselon; M.V. Gorshkov (433-440).
The combination of liquid chromatography (LC) with mass spectrometry (MS) has become a mainstream proteome analysis strategy. In LC–MS, measured masses possess their “universal” scale derived from atomic mass tables. In contrast, the observed LC retention times (RT) are not tied to a conventional time scale, and depend on experimental conditions. However, RT data, being explicitly orthogonal to MS, offer relevant information for proteome characterization. We present here a strategy for peptides RT data standardization, based on the generation of a standard scale using retention prediction models, which enables sharing of identification databases in the context of multi-laboratory research.
Keywords: Reversed phase liquid chromatography; Retention time correlation; Proteomics; AMT tag; Mass spectrometry;

Simultaneous liquid chromatographic analysis of ritonavir, quinine and 3-hydroxyquinine in human plasma by Julius O. Soyinka; Cyprian O. Onyeji; Sharon I. Omoruyi (441-445).
In regions with high prevalence of HIV and malaria, co-infection of both diseases is common; hence, there is a high possibility of concurrent administration of antiretroviral and antimalarial drugs. This study describes a new ion-pair reversed-phase high-performance liquid chromatographic (HPLC) method for simultaneous determinations of ritonavir (RTV), quinine (QN), and its major metabolite, 3-hydroxyquinine (3-HQN), in human plasma. Following a simple extraction with diethyl-ether under alkaline conditions, chromatographic separation was achieved on a 5-μm particle size C-18 column (200 mm × 4.6 mm I.D.) using a mobile phase consisting of methanol:acetonitrile:0.02 M potassium dihydrogen phosphate (15:10:75) containing 75 mmol/L perchloric acid (pH 2.8). Retention times for RTV, 3-HQN, QN and the internal standard were 2.8, 4.0, 7.0 and 12 min, respectively. The limits of detection and validated lower limits of quantitation were 10 and 12.5 ng/ml for RTV while the corresponding values were 5 and 70 ng/ml for both QN and 3-HQN, respectively. The new HPLC method is simple, rapid, selective, reproducible and cost-effective. As demonstrated in three volunteers, it will facilitate the conducting of simultaneous therapeutic monitoring of quinine and ritonavir in patients concurrently receiving both drugs.
Keywords: Ritonavir; Quinine; 3-hydroxyquinine; Liquid chromatography;

Plasma hydroxyurea determined by gas chromatography–mass spectrometry by Tayeb Kettani; Frédéric Cotton; Béatrice Gulbis; Alina Ferster; Alain Kumps (446-450).
Hydroxyurea treatment is efficiently used to ameliorate the clinical course of patients affected with sickle cell disease. To understand the patient's wide variation in the clinical response to that drug and monitor its plasma levels, a new method was developed and validated. Fifty μL plasmatic samples containing hydroxyurea are added with internal standard, deproteinized, evaporated to dryness, silanized, and analyzed by gas chromatography–mass spectrometry, which operates in the selected ion mode after electron impact fragmentation. Linearity was found to extend to at least 100 mg/L. Over a 1–25 mg/L concentration range, coefficients of variation for intra-day and inter-day precision are 5.3% and 7.7%, respectively. Plasma blank-samples reveal endogenous hydroxyurea at a level ≤0.2 mg/L. The performances of the method, which is fast and simple, encounter the analytical goals needed for evaluation of hydroxyurea treatment and for pharmacokinetic studies.
Keywords: Hydroxyurea; Gas chromatography–mass spectrometry; Plasma; Pharmacokinetics; Sickle cell disease;

Enzymatic diagnosis of Sjögren-Larsson syndrome using electrospray ionization mass spectrometry by Robert-Jan Sanders; Rob Ofman; Conny Dekker; Stephan Kemp; Ronald J.A. Wanders (451-455).
Sjögren-Larsson syndrome is a metabolic disorder characterized by accumulation of long-chain fatty alcohols in plasma of patients due to mutations in the ALDH3A2 gene, that codes for a microsomal fatty aldehyde dehydrogenase (FALDH). Recent studies have demonstrated that FALDH is involved in the last step of the conversion of 22-hydroxy-C22:0 into the dicarboxylic acid of C22:0 (C22:0-DCA).FALDH activity was determined by incubating fibroblast homogenates with ω-hydroxy-C22:0 in the presence of NAD+. Electrospray ionization mass spectrometry (ESI-MS) was used to quantify the amounts of C22:0-DCA produced.All SLS patients were deficient in C22:0-DCA productions with activities ranging from 3.2–26.3% of mean control.The new assay described in this paper has substantial advantages over previous assays, and allows for the easy, reliable and rapid diagnosis of SLS.
Keywords: SLS; ALDH3A2; Sjögren-Larsson syndrome; FALDH; ESI-MS;

A fast, simple, and sensitive sample preparation procedure based on dispersive liquid–liquid microextraction (DLLME) is proposed for the determination of cholesterol in food samples using isocratic reverse phase high performance liquid chromatography (RP-HPLC) and UV detection. The influence of several important parameters on extraction efficiency of cholesterol was evaluated. Under optimized conditions, a linear relationship was obtained between the peak area and the concentration of cholesterol in the range of 0.03–10 μg l−1. The detection and quantification limits were 0.01 and 0.03 μg l−1, respectively. Intra-day and inter-day precisions for the analysis of cholesterol were in the range of 1.0–3.1%. The applicability of the proposed method was demonstrated by analyzing cholesterol in milk, egg yolk and olive oil.
Keywords: Disperser liquid–liquid microextraction; Cholesterol; Milk; Yolk; Olive oil; HPLC;

We present a rapid and simple method for detecting novobiocin in biologic samples using a methanol-based extraction of the tissue matrix and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. The sample, prepared using centrifugal ultrafiltration with 5.0% SDS, was directly injected into the LC-MS/MS. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.5% formic acid in water/methanol. The method was validated according to the Japanese Maximum Residue Limits recommendations. Detection was linear over a range of 5–100 ppb matrix solution (r  > 0.998). Novobiocin recovery values from chicken (0.05 ppm) and fish tissues (0.05 ppm), milk (0.08 ppm), and human serum (0.05 and 0.01 ppm) samples ranged from 71 ± 1 to 95 ± 2%.
Keywords: Novobiocin; Chicken tissue; Fish tissue; Milk; Human serum; Centrifugal ultrafiltration; Liquid chromatography tandem mass spectrometry;