LC–MS/MS Method for the Simultaneous Determination of Free Urinary Steroids

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC–MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap^® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1–120 ng/mL for cortisol and cortisone, and 1–120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85–105 %, respectively. Our LC–MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11β-HSD2 and 11β-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.     

Coupled Turbulent Flow Chromatography: LC–MS/MS Method for the Analysis of Pesticide Residues in Grapes, Baby Food and Wheat Flour Matrices

This paper describes an on-line sample preparation method for the simultaneous determination of 48 pesticides in grapes, baby food and wheat flour matrices. Target pesticides were selected to represent a wide variety of chemical structures and three typical matrices were selected. Turbulent flow chromatography was applied for on-line sample cleanup directly coupled to LC?CMS/MS. The aim of the method was to reduce total analysis time, eliminate manual laboratory work, provide clean extracts and achieve reproducible results. Single laboratory method validation was conducted establishing limits of detection between 0.8 and 6.0?ng?g^?1 for baby food, and 0.8?C10.3?ng?g^?1 for other matrices. Within-day precision values varied between 4 and 18?%, while between-day precisions were in the range 5?C22?%. Method recovery ranged from 67 to 124?%, and method accuracy was demonstrated by analysis of external quality control samples. The method was also tested on 24 different survey samples from both bio and organic origin. The method was shown to be convenient, fast and fit for purpose in meeting regulatory requirements for pesticide residue monitoring.     

Coupled Turbulent Flow Chromatography: LC–MS/MS Method for the Analysis of Pesticide Residues in Grapes,Baby Food and Wheat Flour Matrices

This paper describes an on-line sample preparation method for the simultaneous determination of 48 pesticides in grapes, baby food and wheat flour matrices. Target pesticides were selected to represent a wide variety of chemical structures and three typical matrices were selected. Turbulent flow chromatography was applied for on-line sample cleanup directly coupled to LC–MS/MS. The aim of the method was to reduce total analysis time, eliminate manual laboratory work, provide clean extracts and achieve reproducible results. Single laboratory method validation was conducted establishing limits of detection between 0.8 and 6.0 ng g⁻¹ for baby food, and 0.8–10.3 ng g⁻¹ for other matrices. Within-day precision values varied between 4 and 18 %, while between-day precisions were in the range 5–22 %. Method recovery ranged from 67 to 124 %, and method accuracy was demonstrated by analysis of external quality control samples. The method was also tested on 24 different survey samples from both bio and organic origin. The method was shown to be convenient, fast and fit for purpose in meeting regulatory requirements for pesticide residue monitoring.

     

LC–MS/MS Method for Simultaneous Determination of Monoethanol- and Dimethylnitramine in Aqueous Soil Extracts

Monoethanol (MEA)- and dimethyl (DMA)-nitramines are by-products of amine-based post-combustion CO₂ capture (PCCC) processing, and are potentially carcinogenic. The compounds are challenging to measure, also with LC–tandem mass spectrometry (MS/MS), attributed to their high polarity and extreme proneness to matrix effects. In contrast to related methods, the MEA- and DMA-nitramines were simultaneously determined in aqueous soil extracts in less than 10 min using a 1 mm × 150 mm Atlantis^® T3 (3 µm) C18 column. A mobile phase of water/methanol (90/10, v/v) and 2 mM acetic acid allowed for electrospray ionization (ESI) of both analytes [in contrast to the need for both ESI and atmospheric pressure chemical ionization (APCI) in related methods]. Polarity switching electrospray was required for the simultaneous detection of the analytes, and concentration limits of detection (LODs) in the aqueous soil extracts were ≤5.0 µg L^?1 using an injection volume of 20 μL and no prior enrichment step. Matrix effects were compensated for using isotope-labelled internal standards, and satisfactory precision and linearity were obtained (within- and between-day precisions ≤19%, r ² ≥ 0.995 for concentrations up to 500.0 µg L^?1). To avoid signal decrease over time when measuring DMA-nitramine alone, the use of polarity switching was beneficial, in addition to frequent cleaning of the ion transfer capillary. The validated method can be used to determine nitramines in aqueous soil extracts, which is of importance as soil sorption is a determinant of the compounds’ environmental fate.     

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C₁₈ column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL^?1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL^?1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.     

Detection of β-methylphenethylamine,a novel doping substance,by means of UPLC/MS/MS

Novel substances of expected doping activity are constantly introduced to the market. β-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid–liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.     

Validated Method to Determine Quetiapine and Norquetiapine in Microsomal Matrix by LC MS–MS: Implication in Quetiapine Metabolism

A sensitive and selective LC–MS/MS method for the quantification of the atypical antipsychotic agent quetiapine and its metabolite norquetiapine (N-desalkyl quetiapine) was developed and validated. Following the protein precipitation technique, the analytes were separated using a reversed phase column with gradient elution. The compounds were ionized in the electrospray positive ionization (ESI+) ion source tandem MS detection in multiple reaction monitoring (MRM) mode. Calibration curves were generated by plotting the peak area ratio of quetiapine and norquetiapine to the IS clozapine for each calibration concentration. The method provides a linear response from a quantitation range of 2.3–452.9 nM (0.9–173.7 ng/mL) and 2.7–543.0 nM (1.0–200.0 ng/mL) for quetiapine and norquetiapine, respectively. Regression analysis showed a correlation coefficient greater than 0.999 and 0.991 for quetiapine and norquetiapine, respectively. To evaluate the metabolism of quetiapine by the cytochrome P450 in microsomes, the method has been subsequently employed. LC–MS/MS procedure has been carried out to determine increasing concentrations of both drugs in microsomal matrix obtained by a pool of mammalian liver microsomes BD UltraPool^TM Human Liver Microsomes (HLM 150).     

LC–MS Method for the Sensitive Determination of Metoclopramide: Application to Rabbit Plasma, Gel Formulations and Pharmaceuticals

To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C₁₈ column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min^?1. The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL^?1 with a limit of detection of 0.077 pg μL^?1 for standard solutions and 2.5–200 pg μL^?1 with a limit of detection of 0.42 pg μL^?1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H₂O₂, HCl and NaOH were also investigated.     

The Rapid Analysis of Antibiotics in Animal Meat and Egg Using a Novel SEP Method and UPLC–MS/MS

A simple and sensitive method for the determination of 39 antibiotics, including sulfonamides and quinolones, in pork, chicken, fish tissues and eggs, has been developed. The sample preparation included ultrasound-assisted extraction (UAE) with 0.1% formic acid in 90:10 acetonitrile/water (v:v) and a final clean-up with Oasis PRiME HLB, a new reversed phase SPE without traditional pre-equilibration and washing steps before eluting SPE. Analysis was performed by ultra-performance liquid chromatography coupled to tandem quadrupole mass spectrometry (UPLC–MS/MS). The positive ionization in multiple reaction monitoring mode (MRM) and product ion confirmation scan (PICs) were used in the method. The PICs provides additional confirmation for compound identification through acquisition of MS/MS spectra in the same injection and a means of verifying that the signal from the MRM peak is from the compound of interest. In particular, single test is simultaneously able to gain both quantitative MRM and qualitative full-scan MS/MS data without the need for long analysis times or repeat injection. All solvent and matrix-matched calibration curves showed excellent correlation coefficient >0.990, with the dynamic range 0.2–100 ng mL^?1. For over 90% of the analytes, the recoveries were between 60 and 120% in all matrices studied at three spiked levels of low, medium, and high concentrations, with the intra-day precision values in the range of 2.7–20.0% and the inter-day precision values in the range of 6.2–21.3%. The limits of detection (LODs) and limits of quantification (LOQs) of all drugs were 0.05–2.6 and 0.12–5.6 μg kg^?1, respectively. A weak matrix effect was observed for most of the compounds in four complex samples. The proposed method was proven very simple, fast, sensitive, and selective and has been successfully applied in real samples from local markets and farms.     

Simultaneous Determination of 16 Illegally Added Drugs in Capsule Dietary Supplements Using a QuEChERS Method and HPLC–MS/MS

Chromatographia - A simple and rapid method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) pretreatment and HPLC–MS/MS (high performance liquid...     

LC–MS–MS Analysis of Strictosamide in Rat Plasma, and Application of the Method to a Pharmacokinetic Study

A rapid and simple high-performance liquid chromatographic tandem mass spectrometric method has been developed and validated for analysis of strictosamide in rat plasma. Chromatographic separation was achieved on a C₁₈ column by gradient elution with mixtures of methanol, water, and acetonitrile containing 0.05% acetic acid. Digoxin was used as internal standard. Selected reaction monitoring (SRM) was used for MS quantitation. Linearity was good in the range 0.05–20 ng mL^?1 in rat plasma. The lower limit of quantitation was 0.04 ng mL^?1. The method is precise and reliable and can be applied to pharmacokinetic studies.     

GC/MS和ESI/MS/MS同位素内标法检测甲基丙二酸血症

以甲基丙二酸血症为对象,分别用GC/MS和ESI/MS/MS方法对该疾病进行了定性和定量检测.通过对样品前处理和分离条件的改善,对疾病的标识化合物之一甲基丙二酸进行了定量测定,其稳定性、精密度和回收率结果很好.同时比较了GC/MS和ESI/MS/MS两种方法的特点,发现两种方法的结合不仅可满足新生儿代谢疾病筛查的要求,同时还可对高危人群进行诊断.     

Comparison of Different Sorbents in the QuEChERS Method for the Determination of Pesticide Residues in Strawberries by LC–MS/MS

A comparison of sample preparation based on the quick, easy, cheap, effective, rugged and safe (QuEChERS) method for analysis of pesticide residues in strawberries by LC–MS/MS was made using different sorbents in the clean-up by dispersive solid-phase extraction (d-SPE). Some sorbents were laboratory-made, prepared by depositing poly(methyloctadecylsiloxane) (PMODS), poly(methyloctylsiloxane) (PMOS), aminopropyl-terminated poly(dimethylsiloxane) (APPS) and copolymer of (52–48 %)dimethyl-(48–52 %)methylphenyl-siloxane (DMMPS) onto silica supports. The commercial sorbent primary–secondary amine (PSA) and mixtures of two sorbents, primary–secondary amine and poly(methyloctadecylsiloxane), were also used in the experiments. The performances of the sorbents were evaluated by parameters such as color of the final extract, gravimetric measurement, recovery and matrix effect at the fortification level of 100 ng g^?1 of the pesticide mixture in strawberries. The recoveries were in the range 70–120 %, and the RSD values were lower than 20 % for most of the pesticides using the modified QuEChERS method with different sorbents in the clean-up step. The strawberry extracts were cleaned more efficiently with the use of primary–secondary amine sorbent, which has the function of removing sugars, organic acids and especially pigments. The sample preparation method was efficient, and LC–MS/MS determination was optimal because of high selectivity and good detectivity for the multiresidue analyses.     

LC/MS/MS方法筛查新生儿苯丙酮尿症

苯丙酮尿症 [1～ 4 ] ( PKU )发病原因是患者基因缺陷使肝脏不能合成苯丙氨酸羟化酶而导致体内苯丙氨酸 ( Phe)不能正常代谢为酪氨酸 ( Tyr) ,前者在体内大量堆积并氧化为对人体有害的苯丙酮酸 . PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,在全世界每年 [5]约有一千万婴儿接受 PKU筛查 ;在我国 ,PKU也是卫生部要求重点筛查的病种 .Chace[5]等在 1 993年报道了 MS/ MS方法筛查新生儿PKU:直接使用 MS/ MS的中性碎片丢失扫描方式检测 Phe和 Tyr,通过氘代内标与待测氨基酸的质谱峰高比来定量 .MS/ MS方法速度快、准确性好、可以…     

Simultaneous Determination of α- and γ-Mangostins in Mouse Plasma by HPLC–MS/MS Method: Application to a Pharmacokinetic Study of Mangosteen Extract in Mouse

Recently, extracts from the pericarp of mangosteen, Garcinia mangostana L., exhibited various pharmacological properties such as anti-oxidative, anti-inflammatory, anti-bacterial and chemopreventive activities. Albeit it has diverse application, there is little information about its pharmacokinetic aspects. Thus, the present study was undertaken to develop the simultaneous determination of α- and γ-mangostins (α- and γ-MG), major and active compounds, from extracts for the application of pharmacokinetic studies in mice using combined liquid chromatography–tandem mass-spectrometry and microsampling systems. The intra- and inter-validation, precision, accuracy, stability, recovery and matrix effects of α- and γ-MG were conducted in mouse plasma. Based on the developed analytical methods, pharmacokinetic parameters of α- and γ-MG after intravenous and oral administration of mangosteen extract were calculated. In sample preparation steps, the biological samples were deproteinized by acetonitrile and chromatographic separation was accomplished on a C₁₈ column. The detection was accomplished by multiple-reaction monitoring scanning after electrospray ionization source in the positive ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 411.062 → 354.900, 397.384 → 340.900, and 808.379 → 527.200 for α- and γ-MG and docetaxel (internal standard), respectively. The total run time was 5 min. The results provided a meaningful basis for the preclinical and clinical application of mangosteen extract.     

LC/MS/MS法测定化妆品中树苔提取物

采用液相色谱-质谱法测定了化妆品中的树苔提取物,建立了化妆品中树苔提取物的检测方法,方法的检出限为10 ng/mL,被测物在10～500 ng/mL范围内线性关系良好.本方法可以用于树苔提取物在化妆品中含量的质量控制.     

Simultaneous Determination of α- and γ-Mangostins in Mouse Plasma by HPLC–MS/MS Method: Application to a Pharmacokinetic Study of Mangosteen Extract in Mouse

Recently, extracts from the pericarp of mangosteen, Garcinia mangostana L., exhibited various pharmacological properties such as anti-oxidative, anti-inflammatory, anti-bacterial and chemopreventive activities. Albeit it has diverse application, there is little information about its pharmacokinetic aspects. Thus, the present study was undertaken to develop the simultaneous determination of α- and γ-mangostins (α- and γ-MG), major and active compounds, from extracts for the application of pharmacokinetic studies in mice using combined liquid chromatography–tandem mass-spectrometry and microsampling systems. The intra- and inter-validation, precision, accuracy, stability, recovery and matrix effects of α- and γ-MG were conducted in mouse plasma. Based on the developed analytical methods, pharmacokinetic parameters of α- and γ-MG after intravenous and oral administration of mangosteen extract were calculated. In sample preparation steps, the biological samples were deproteinized by acetonitrile and chromatographic separation was accomplished on a C₁₈ column. The detection was accomplished by multiple-reaction monitoring scanning after electrospray ionization source in the positive ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 411.062 → 354.900, 397.384 → 340.900, and 808.379 → 527.200 for α- and γ-MG and docetaxel (internal standard), respectively. The total run time was 5 min. The results provided a meaningful basis for the preclinical and clinical application of mangosteen extract.

     

A Simple and Sensitive LC–MS/MS Method for Simultaneous Determination of Temsirolimus and Its Major Metabolite in Human Whole Blood

A simple, fast and sensitive LC?CMS/MS method was developed and validated for the simultaneous determination of the concentrations of temsirolimus and its major metabolite, sirolimus, in human whole blood. The blood sample (100???L) after adding temsirolimus-d7 and sirolimus-d3 internal standards was precipitated with 0.200?mL of methanol/0.300?M zinc sulfate (70/30, v/v), then analyzed by a Shimatzu LC system coupled to a Sciex API-5000 mass spectrometer. The chromatographic separation was carried out on a BDS Hypersil C8 column (50?×?3.0?mm, 5???m) at 50?°C with a mobile phase composed of methanol/water/formic acid (72/28/0.1) (v/v/v) containing 2.50?mM ammonium acetate. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode. This method was validated from 0.250 to 100?ng?mL^?1 for temsirolimus and 0.100 to 40.0?ng?mL^?1 for sirolimus. The lower limits of quantitation were 0.25?ng?mL^?1 for temsirolimus and 0.1?ng?mL^?1 for sirolimus. The intra-day and inter-day precisions (CV?%) of spiked quality control (QC) samples were less than 10.4 and 9.6?%, respectively. The accuracies as determined by the relative error for QC samples were less than 12.1?% for intra-day and 7.3?% for inter-day. No significant matrix effect was observed. This method has been successfully applied to analyze clinical pharmacokinetic study samples. The assay reproducibility was also demonstrated by using incurred samples.