Journal of Chromatography B (v.871, #1)

n-Octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monoliths were prepared for rapid screening, determination and one-step purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata). The modified monolith showed a specific surface area of 17.8 m2  g−1, an average pore size of 0.76 μm and a total porosity of 60.8%. Fast separation of R. puerariae crude extract was achieved within 5 min at a flow velocity of 722 cm h−1 resulting in a puerarin purity of 97%, with a recovery of 85%. This demonstrates the potential of n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith for the rapid analysis and separation of isoflavonoids. Preparative scale sample loading (12 mg in 2 mL) resulted in a purity of 95%, and a recovery of about 69%. HPLC, FTIR, MS and 1H NMR spectroscopy were used for the characterization and quantification of puerarin in isolated fraction.
Keywords: Monolith; Chromatography; Radix puerariae; Puerarin; Isoflavonoids;

Metabolic analysis of four phenolic acids in rat by liquid chromatography–tandem mass spectrometry by Zi-chuan Zhang; Man Xu; Shi-feng Sun; Xue Qiao; Bao-rong Wang; Jian Han; De-an Guo (7-14).
A liquid chromatography-diode array detection-electrospray ionization ion trap mass spectrometry (LC–DAD–ESI-MS n ) method was established for the analysis of danshensu, caffeic acid, ferulic acid and isoferulic acid in rat plasma, bile, urine and feces after oral administration or intravenous injection. Liquid–liquid extraction was employed for the preparation of biosamples, and the chromatographic separation was carried out using an Agilent Zorbax Extend C18 reversed phase column and acetonitrile-0.1% formic acid as the mobile phase. Totally nineteen metabolites were detected and identified as prototype, methylated, hydroxylated, sulfated and glucuronized conjugates. The metabolism of the individual phenolic acids in biosamples was investigated, and the metabolic pathway was proposed. By comparing the metabolism of different compounds which shared similar structures, we were able to find that methylation was the main pathway of danshensu metabolism, and the double bond on the side chain was critical for the drug excretion via bile and the formation of glucuronized conjugates. The results proved that the established method was simple, sensitive and reliable, which could be used to detect and identify the structures of metabolites and to better understand their in vivo metabolism.
Keywords: Danshensu; Caffeic acid; Ferulic acid; Isoferulic acid; Phenolic acid; Metabolism; HPLC–MS;

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 μL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm × 2.1 mm Agilent Zorbax SB-phenyl 5 μm column coupled to a 3 mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 μL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3 > 541.3, 544.2 > 501.2, and 615.3 > 572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5–3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 μL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.
Keywords: 17-(Allylamino)-17-demethoxygeldanamycin; 17AAG; 17-(Amino)-17-demethoxygeldanamycin; 17AG; HSP90; LC–MS–MS;

Three protocols for fatty acid analysis in Sinorhizobium meliloti were improved by the addition of a number of standards/controls and a silylation step which allowed the determination of recoveries, extents of conversion of lipids to fatty acid methyl esters (FAMEs) and extents of side reactions. Basic hydrolysis followed by acid-catalyzed methylation and transmethylation with sodium methoxide, were the best for the analysis of 3-hydroxy- and cyclopropane fatty acids, respectively. A micro-scale, one-vial method that employed sodium methoxide/methanol was equally efficient and on a 1000-fold smaller scale than standard methods. Because this method avoids aqueous extractions, 3-hydroxybutanoic acid was detected as its trimethylsilyloxy methyl ester along with FAMEs.
Keywords: Fatty acid methyl esters; Sinorhizobium meliloti; Cyclopropane fatty acids; 3-Hydroxy fatty acids; 3-Hydroxybutanoic acid; Poly(3-hydroxybutanoate); Hydrolysis;

A simple and reliable method for determining plasma concentration of dehydroxymethylepoxyquinomicin by high performance liquid chromatography with mass spectrometry by Etsuko Watanabe; Nobuo Mochizuki; Hidetomo Ajima; Keiko Ohno; Mitsuhiro Shiino; Kazuo Umezawa; Moto Fukai; Michitaka Ozaki; Hiroyuki Furukawa; Satoru Todo; Satoshi Kishino (32-36).
We have developed a simple and reliable method for determining plasma concentration of dehydroxymethylepoxyquinomicin (DHMEQ), a new low molecular weight NF-κB inhibitor, using high performance liquid chromatography with mass spectrometry (LC–MS). An experiment of mass spectrometry with electrospray ionization in the negative ionization mode was performed to detect ion transitions at m/z 260.05 [M−H] for DHMEQ and 240.29 for mefenamic acid as an internal standard. The samples were purified using liquid–liquid extraction with ethyl acetate. The method yielded a standard curve which was linear for the concentration range of 0.1–125 ng/mL when 0.05 mL plasma was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 50 pg/mL (signal/noise >3). Daily fluctuation of plasma standard curve was small. The intra- and inter-assay precision ranged from 2.84 to 4.76% (n  = 6) and 2.91 to 7.03% (n  = 6), respectively. The LC–MS technique described provides a simple and reliable liquid chromatographic method for the determination of DHMEQ level and for use in studies involving pharmacokinetics.
Keywords: Dehydroxymethylepoxyquinomicin; DHMEQ; HPLC; Mass spectrometry;

LC–MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: Optimization of pre-LC–MS procedure parameters by Ruben t’Kindt; Lieven De Veylder; Michael Storme; Dieter Deforce; Jan Van Bocxlaer (37-43).
This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC–ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC–MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC–MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H2O 80/20. As such, an efficient and highly reproducible LC–MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC–MS (8.88% ± 5.16 versus 7.05% ± 4.45) and technical variability (12.53% ± 11.21 versus 9.31% ± 6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.
Keywords: Metabolite extraction; Mass spectrometry; Micro-liquid chromatography; Plant metabolomics; Pre-LC–MS treatment;

A precise, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine (mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 μL aliquots of human plasma via liquid–liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm × 2.1 mm, 5 μm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/z 372.2 → 176.2, 197.2 → 118.1 and 470.5 → 274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0–3000.0 ng/mL for TRZ and 0.2–60.0 ng/mL for mCPP was evaluated with mean correlation coefficient (r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was ≤8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100 mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.
Keywords: Trazodone; m-Chlorophenylpiperazine; Human plasma; LC–MS/MS; Liquid–liquid extraction;

Haemoglobin Noah Mehmet Oeztuerk (α2 δ2143 (H21)His → Tyr: A novel δ-chain variant in the 2,3-DPG binding site by Emmanuel Bissé; Christine Schaeffer; Agnès Hovasse; Sabine Preisler-Adams; Thomas Epting; Manfred Baumstark; Alain Van Dorsselaer; Jürgen Horst; Heinrich Wieland (55-59).
A new δ-chain variant, δ143 (H21) His → Tyr or Hb Noah Mehmet Oeztuerk, was discovered during the investigation of the cause of hemolytic anaemia in a 6-month-old infant of Turkish descent. It was detected by Cation exchange high-performance liquid chromatography (CE-HPLC) using PolyCAT A column. P 50 was 20.6 ± 0.60 mmHg and 29.3 ± 0.40 mmHg for the carrier and the wild-type, respectively. This suggests an increase in oxygen affinity. On routine CE-HPLC Hb A2 was low (1.2%) and the variant was not detected. An extended family study revealed that the variant was not associated with the anaemia or with any other clinical abnormality.
Keywords: Hb δ-variant; Nano-LC–MS; HPLC; Oxygen affinity;

A biospecific lectin-affinity-based isolation process for a novel glycoprotein (ClGp1) from the venom of the pelagic jellyfish Cyanea lamarckii, is described and the isolated glycoprotein is chemically and biologically characterized according to size, molecular interaction and toxicity. The molecular mass of the isolated protein is 25.7 kDa as determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF). The carbohydrate content was calculated after enzymatic deglycosylation as 6.85 kDa. The glycoprotein is cytotoxic and could be isolated from cnidocysts of mesenteric and fishing tentacles. The binding behaviour of the glycoprotein to the lectins Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) was analyzed by surface plasmon resonance (SPR) and affinity constants in the range of K D  = 3.0 × 10−7  M for ConA and 2.1 × 10−6  M (pH 5.0) and 2.6 × 10−6  M (pH 7.4) for WGA were obtained.
Keywords: Glycoprotein; SPR; Affinity chromatography; Lectin; Cyanea;

A novel-immobilized enzyme strategy created by magnetic nanospheres for monitoring enzyme activity and screening inhibitors followed by high performance liquid chromatography (HPLC) has been demonstrated. Through the reaction of the aldehyde groups with amine groups, α-glycosidase was simply and stably immobilized onto magnetic nanospheres by the cross-linking agent glutaraldehyde. In order to profiling the activity of the immobilized α-glucosidase, the natural substrate was hydrolyzed by it and the yield of product was determined by HPLC. Compared with traditional bioassay approach, the prepared immobilized α-glucosidase displays a high activity and stability which allows it to be easily reused for 10 times. Enzyme inhibition assays by known inhibitor glucobay and three candidate traditional Chinese medicines (TCMs) were then investigated using a similar methodology. This assay was able to readily detect the change of the immobilized enzyme activity based on measuring a decrease of product formation using HPLC. The approach is general and offers many attractive advantages including easy product isolation, inexpensive cost, and high efficiency in terms of reagent consumption.
Keywords: Enzyme immobilization; Magnetic nanospheres; α-Glucosidase; Liquid chromatography; Enzyme inhibitor; Traditional Chinese medicine;

A LC–MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE2, that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d6) analog followed by LC–MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC–MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.
Keywords: PGE2; Tetranor PGE-M; Tetranor PGA-M; Creatinine; Human; Urine; Aspirin; Prostaglandins; LC–MS/MS; Isotope dilution method;

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4′-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1′-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1′-hydroxymidazolam), ofloxacin (for 4′-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis® HLB cartridges. The chromatography was performed using a C18 column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple–quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1′-hydroxymidazolam, brodimoprim, 4′-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5–2500 ng/mL for phenacetin, 2.5–2500 ng/mL for paracetamol, 5–500 ng/mL for midazolam, and 0.5–500 ng/mL for 1′-hydroxymidazolam. In urine calibration ranges were 5–1000 ng/mL for dextromethorphan, 0.05–10 μg/mL for dextrorphan and 4′-hydroxymephenytoin, 5–2000 ng/mL for tolbutamide, 0.05–20 μg/mL for 4-hydroxytolbutamide and 0.025–10 μg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3–12.4% and 1.5–14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from −9.1 to 8.3% and −10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1′-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4′-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, −20 °C), three freeze–thaw cycles and autosampler (24 h, 4 °C) stability. The stability of urine samples was also prepared with and without β-glucuronidase incubation (37 °C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.
Keywords: Cytochrome P450; LC–MS/MS; Phenotyping; In vivo; Rat;

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the determination of allylestrenol in human plasma was established. Plasma samples were extracted by tert-butyl ether and separated by LC/MS/MS using a Phenomenex Curosil-PFP column (250 mm × 4.6 mm ID, dp 5 μm) with a mobile phase of methanol–water (95:5, v/v). The analytes were monitored with atmospheric pressure chemical ionization (APCI) by selected reaction monitoring (SRM) mode. The linear calibration curves covered a concentration range of 0.04–20.0 ng/mL with lower limit of quantification (LLOQ) at 0.04 ng/mL. The mean extraction recovery of allylestrenol was greater than 81.8%. The intra- and inter-day precisions were less than 1.3% and 3.1% respectively, determined from quality control (QC) samples of three representative concentrations. The method has been successfully applied to determining the plasma concentration of allylestrenol and a clinical pharmacokinetics study in healthy Chinese female volunteers.
Keywords: Allylestrenol; Pharmacokinetics; Mifepristone; LC/MS/MS;

Tetrodotoxin poisoning evidenced by solid-phase extraction combining with liquid chromatography–tandem mass spectrometry by Hsiao-Chin Jen; Shin-Jung Lin; Yung-Hsiang Tsai; Chun-Hsiang Chen; Zu-Chun Lin; Deng-Fwu Hwang (95-100).
The toxicity and toxin component of gastropod Niotha clathrata implicated to a food paralytic poisoning incident in Kaohsiung, Taiwan in November 2006 were studied. The highest scores of average toxicity in the digestive gland and other portions from collected gastropods were 62 ± 24 (mean ± S.D.) and 32 ± 16 μg/g according to tetrodotoxin (TTX) bioassay, respectively. The toxin from these gastropods was large amount and easily identified as tetrodotoxin by traditional method of HPLC-FLD. The toxin of patient's blood serum was trace amount and analyzed by a new developed method LC–MS/MS. LC–MS/MS was contracted by the LC system interfaced with the MS/MS system with a turbo ion spray interface. Positive ion detection and multiple reaction monitoring mode were used for TTX of patient serum. It was found that linearity in serum was observed within concentration ranged of 1–100 ng/ml and limit of detection was 0.1 ng/ml. The LOQ was reproducible at 1 ng/ml in serum. The blood serum showed to contain TTX of 3.30 ± 0.08 ng/ml. It indicated that LC–MS/MS was more lower detectable and believable method for TTX determination than LC–MS reported previously. Furthermore, the causative agent of gastropod food poisoning was identified as TTX.
Keywords: Tetrodotoxin; Niotha clathrata; HPLC; LC–MS/MS; Blood;

A direct liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for measurement of urinary Δ9-tetrahydrocannabinol carboxylic acid (THCA) was developed. The method involved dilution of the urine sample with water containing 2H9-deuterated analogue as internal standard, hydrolysis with ammonia, reversed phase chromatography using a Waters ultra-performance liquid chromatography (UPLC™) equipment with gradient elution, negative electrospray ionization, and monitoring of two product ions in selected reaction monitoring mode. The measuring range was 2–1000 ng/mL for THCA, and the intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 5%. Influence from urine matrix on ionization efficiency was noted in infusion experiments, but was compensated for by the internal standard. Comparison with established gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry methods in authentic patient samples demonstrated accuracy in both qualitative and quantitative results. A small difference in mean ratios (∼15%) may be explained by the use of different hydrolysis procedures between methods. In conclusion, the high efficiency LC–MS/MS method was capable of accurately identify and quantify THCA in urine with a capacity of 14 samples per hour.
Keywords: Urine; Drug testing; (±)-11-Nor-9-carboxy-Δ9-tetrahydrocannabinol; LC–MS/MS;

This work investigated the spectrum-effect relationships between HPLC fingerprints and the anti-bacterial activities of EtOAc extracts from Radix Isatidis. Fingerprints of EtOAc extracts of Radix Isatidis from various sources were established by a High-Performance Liquid Chromatography. The process of Escherichia coli (E. coli) growth affected by EtOAc extracts was monitored using a Thermal Activity Monitor (TAM) Air Isothermal Calorimeter by microcalorimetry. By analyzing the power–time curves, quantitative parameters, such as growth rate constant k, maximum heat-production rate P m, appearance time t and total heat-production Q were obtained to characterize the interactions of E. coli and the EtOAc extracts from Radix Isatidis. The HPLC fingerprints were investigated using hierarchical clustering analysis. The main thermo-kinetic parameters from the power–time curves were analyzed using principal component analysis. The spectrum-effect relationships between the HPLC fingerprints and anti-bacterial activities were analyzed with multivariant correlation analysis. Close correlation existed between the spectrum-effect relationships of the EtOAc extracts. Salicylic acid in the HPLC fingerprints might be one of the anti-bacterial components. This work provides a general model of the combination of HPLC and microcalorimetry to study the spectrum-effect relationships of EtOAc extracts from Radix Isatidis, which can be used to search for principal components of Radix Isatidis on bioactivity.
Keywords: Radix Isatidis; HPLC fingerprint; Anti-bacterial activity; Microcalorimetry; Spectrum-effect relationship;

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the β2 adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC–MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.
Keywords: LC–MS/MS; Nucleotides; Metabolite profiling; Chinese hamster ovary cells;

Automated two-dimensional liquid chromatography using the PF2D system from Beckman Coulter provides a fractionation platform well suited for differential proteomic studies. To date, the reliability and reproducibility of PF2D has not been accurately tested. Here, we used an optimized software and a pressure-resistant pH electrode, allowing a precise and reproducible control of the pH limits for each fraction during PF2D. We tested the reliability of this improved system by performing several rounds of fractionation using the same protein extract. Three UV maps were generated, leading to 54 chromatograms and more than 3000 protein peaks. Using semi-automated software for peak-to-peak comparison between 2D-LC chromatograms, we demonstrate that the peak concordance is very high. The rates of concordance were higher in the second dimension repeatability tests, indicating that the limiting factors for 2D-LC reproducibility rely on the pI fractionation and sample preparation steps. The reproducibility between maps was closely related to pH curves similarities, further stressing the need of careful pH adjustment and precise electrode calibration.
Keywords: Two-dimensional liquid chromatography; HPLC; Proteomics; Reproducibility; Peak concordance; Chromatogram comparison; PF2D; Chromatofocusing; Reverse phase high-performance liquid chromatography; Non-porous silica;

A simplified method to determine clarithromycin concentrations in human plasma using protein precipitation in a 96-well plate and liquid chromatography–tandem mass spectrometry was developed and validated. Plasma proteins were precipitated with acetonitrile and roxithromycin was used as the internal standard. After vortex mixing and centrifugation, the supernatants were directly injected onto a Phenomenex Luna Phenyl–Hexyl column (50 mm × 2.0 mm ID, 3 μm). The mobile phase consisted of water and methanol (30:70, v/v) containing 0.1% formic acid and 5 mM ammonium acetate. The flow rate was 0.22 mL/min and the total run time (injection to injection) was less than 3 min. Detection of the analytes was achieved using positive ion electrospray tandem mass spectrometry in selected reaction monitoring (SRM) mode. The linear standard curve ranged from 100 to 5000 ng/mL and the precision and accuracy (inter- and intra-run) were within 7.9% and 4.9%, respectively. The method was successfully used to determine clarithromycin concentrations in human plasma samples obtained from healthy subjects who were given clarithromycin 500 mg for 3 days. The method is rapid, simple, precise and directly applicable to clarithromycin pharmacokinetic studies.
Keywords: Clarithromycin; Roxithromycin; Protein precipitation; LC–MS; Human plasma;

Simultaneous analysis of 17α-estradiol and 17β-estradiol in bovine serum by liquid chromatography–tandem mass spectrometry by Giovanni Ferretti; Carolina Ferranti; Teresa Crovella; Maurizio Fiori; Cinzia Civitareale; Camilla Marchiafava; Fernanda delli Quadri; Paolo Cammarata; Luca Palleschi (135-140).
A new LC–MS/MS method for the separation, identification and quantification of residues of 17α-estradiol (17α-E2) and 17β-estradiol (17β-E2) in bovine serum is reported. Deuterium-labelled 17β-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M−H], were generated at m/z 271 and 274 for 17α/17β-E2 and 17β-E2-d 3, respectively. The decision limits obtained (CCα, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17α-E2 and 17β-E2. Detection capability (CCβ, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17α-E2 and 17β-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.
Keywords: Estrogens; α- and β-estradiol; Bovine serum; Liquid chromatography–tandem mass spectrometry;