Ion Spray Interface Research Articles

Liquid Chromatographic-Electrospray Ionization-Mass Spectrometry Method for the Simultaneous Determination of Gramicidin, Neomycin & Triamcinolone Acetonide and Characterization of Novel Forced Degradation Products

The dosage form iotrim (gramicidin, neomycin and triamcinolone) is a combination of two antibiotics (gramicidin and neomycin) and a steroid (triamcinolone). The antibiotics work by killing the bacteria that cause infections. The steroid blocks the action of chemical messengers (prostaglandins) that make the affected area red, swollen and itchy. Consequently, there was still a need to develop a simple, less time consuming and economical method for the simultaneous determination of gramicidin, neomycin and triamcinolone acetonide. The current work is an effort to develop a fast and reproducible LC-MS technique for the simultaneous estimation of gramicidin, neomycin and triamcinolone acetonide. The objective of the present procedure was to validate and develop a precise and accurate liquid chromatography-mass spectrometry (LC-MS) technique for the simultaneous quantification of gramicidin, neomycin and triamcinolone acetonide. Gramicidin, neomycin and triamcinolone acetonide were monitored on Shimadzu-8045 mass spectrometer equipped with electro spray ionization interface. The retention times of gramicidin, neomycin and triamcinolone acetonide were found at 9.145 min, 7.273 min and 2.435 min, respectively. The limit of detection (LOD) results for gramicidin, neomycin and triamcinolone acetonide were observed to be 0.15, 1.5 and 0.6 μg/mL, respectively while the limit of quantification (LOQ) results were observed to be 0.5, 5, 2 μg/mL concentration, respectively. The linear range for gramicidin, neomycin and triamcinolone acetonide were found in the concentration ranges from 1.25-7.5 μg/mL, 12.5-75 μg/mL and 5-30 μg/mL with regression coefficient of 0.9991, 0.9996, 0.9999, respectively. Accuracy values for gramicidin, neomycin and triamcinolone acetonide were found to be in the range of 98.64%, 99.4%, 99.5% respectively. The % RSD for six replicates in precision was less than 2%. According to ICH Q2(R1) recommendations, this method was successfully tested with LC-MS to confirm the chemical structures of newly produced degradation products of triamcinolone acetonide and neomycin. The developed process was validated efficaciously as per ICH guidelines.

Determination of the radical-scavenging activities and identification of anthocyanins from Hexalobus monopetalus ripe fruits

A wild fruit from classified forest of Dindéresso was analyzed for total phenolics, flavonoids, anthocyanins compounds using the Folin-Ciocalteu reagent, spectrophotometric method of Zhishen and colleagues and by the differential pH method respectively. Free radical-scavenging activities of studied fruits extracts were estimated using diammonium 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) salt method. Three major anthocyanins were identified using high performance liquid chromatography coupled with spray ionization interface mass spectrometry. Three identified anthocyanins in fruit were reported to be cyanidin 3-O-(p-coumaroyl) glucoside, pelargonidin 3-O-glucoside and pelargonidin 3-O-rutinoside. In addition, H. monopetalus fruit contained of about 1165±3.1 mg of GAE per 100 g of fresh fruit, 4490±20.2 mg of QE per 100 g of fresh fruit, and 36±0.17 mg of cyanidin 3-O-glucoside equivalents per 100 g of fresh fruit. Total anthocyanin extract had an EC50 = 4.24 mg per mL and a TEC50 time of 21 minutes (intermediate reaction). This free radical-scavenging activity was very low compared to those of the references used (0.024 and 0.034 mg/mL respectively for ascorbic acid and Trolox). The low antiradical activity and reactivity of the H. mucronata extract could be explained by several factors. In any case, fruits of this species were potential sources of natural bioactive substances having beneficial effects on the health of consumers.

Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2’-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2’-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2’-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10–10,000 ng/mL for plasma, and 1–50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.

Fast and Straightforward Method for the Quantification of Pazopanib in Human Plasma Using LC-MS/MS.

Pazopanib is an angiogenesis inhibitor approved for renal cell carcinoma and soft-tissue sarcoma. Studies indicate that treatment with pazopanib could be optimized by adapting the dose based on measured pazopanib plasma concentrations. We describe the validation and clinical application of a fast and straightforward method for the quantification of pazopanib in human plasma for the purpose of therapeutic drug monitoring and bioanalytical support of clinical trials. Stable isotopically labeled C,H3-pazopanib was used as internal standard. Plasma samples were prepared for analysis by protein precipitation using methanol and diluted with 10 mmol/L ammonium hydroxide buffer. Chromatographic separation was performed on a C18 column using isocratic elution with ammonium hydroxide in water and methanol. For detection, a tandem mass spectrometer, equipped with a turbo ion spray interface was used in positive ion mode at m/z 438 → m/z 357 for pazopanib and m/z 442 → m/z 361 for the internal standard. Final runtime was 2.5 minutes. All validated parameters were within pre-established limits and fulfilled the FDA and EMA requirements for bioanalytical method validation. After completion of the validation, the routine application of the method was tested by analyzing clinical study samples that were collected for the purpose of therapeutic drug monitoring. In conclusion, the described method was successfully validated and was found to be robust for routine application to analyze samples from cancer patients treated with pazopanib.

Qualification and Application of a Liquid Chromatography-Tandem Mass Spectrometric Method for the Simultaneous Quantification of Triflusal and its Active Metabolite Desacetyl Triflusal in Human Plasma Using Solid Phase Extraction Technique

Background: Triflusal and Desacetyl Triflusal prevents the formation of blood clots by inhibiting the enzyme cyclooxygenase, and reducing production of thromboxane A2 which is a stimulator of platelet aggregation and thereby reduces the risk of strokes, heart attacks or other serious circulatory problems. Introduction: A rapid, sensitive, specific, accurate, isocratic reversed-phase liquid chromatography coupled with triple quadrapole mass spectrometry method in its tandem operation mode was developed and validated for the simultaneous quantification of Triflusal and Desacetyl Triflusal, in human plasma. Method: Salicylic Acid and Hydrochlorothiazide are used as internal standards. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 µm polymeric sorbent. The reconstituted samples were chromatographed on an Oyster ODS3 100 x 4.6mm, 5µ column using a mobile phase composed of acetonitrile 40:60 (v/v) - 2mM ammonium acetate solutions (pH 3.25 ± 0.3) at a flow rate of 3.2 mL/min. The detection of precursor and product ions of analytes and their internal standards was performed by multiple reaction monitoring mode via a turbo ion spray interface in negative mode by MDS SCIEX API-3000. Result: The calibration curves obtained were linear over the concentration range of 0.100 to 35.086 µg/mL for Triflusal, 1.00 to 120.06 µg/mL for Desacetyl Triflusal with a correlation coefficient r ≥ 0.9952. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. The intra and inter batch precision was ≤ 12.7 for Triflusal and Desacetyl Triflusal with accuracy (%RE) between -6.6 and 6.9. Stability testing showed that all analytes were stable in plasma at 25 °C for 17.83 h, seven freeze–thaw cycles, and at 70°C for 53.65 days. The analytical method was successfully applied to an in vivo study evaluating the pharmacokinetics of Triflusal and Desacetyl Triflusal following single dose oral administration in 24 healthy volunteers in an open label, twoperiod, two sequence, randomized, crossover protocol under fasting conditions. Conclusion: Based on the 90% confidence interval of the individual ratios for Cmax and AUC0-inf, it was concluded that the method is efficient with a very short running time of 3.2min, sufficiently sensitive and suitable for pharmacokinetic studies. Keywords: Liquid chromatography, mass spectrometry, analysis, triflusal, desacetyl triflusal, solid phase extraction, pharmacokinetics, bioavailability.

Development and validation of a liquid chromatography/tandem mass spectrometric method for determination of ethinyl estradiol in human plasma

Introduction: A simple and rapid bioanalytical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on solid phase extraction (SPE) followed by liquid-liquid extraction (LLE) has been developed and validated for quantification of Ethinyl Estradiol in human plasma. Methods: API 5500 LC-MS/MS system with turbo ion-spray interface equipped with pumps (Shimadzu LC-20ADVP), an auto Sampler (Shimadzu SIL-HTC), analytical column SB C18 HT (50 x 3.0 mm, 1.8 μ) and data acquisition system and quantitation program (Applied Biosystems Analyst Software version 1.5, Thermo scientific) was used. The Positive ions were measured in MRM mode for the analyte and Ethinyl Estradiol-d4 used as an internal standard. A composition of 2 mM ammonium formate buffer: acetonitrile (20: 80 v/v) was used as mobile phase. The total run time was 4.0 min. The proposed method has been validated with in the linear range of 5.000–308.560 pg/ml for Ethinyl Estradiol. Results: The retention times of Ethinyl Estradiol and Ethinyl estradiol-d4 were 3.42 min ± 0.30 min and 3.45 min ± 0.30 min respectively. The intraday and interday precision values were within 1.58% to 10.86% and 4.62% to 19.74% respectively for Ethinyl estradiol. The overall recovery for Ethinyl estradiol was found to be 86.91%-103.15%. Conclusion: The method was validated as per ICH guidelines and it would be useful for bioequivalence and pharmacokinetic studies of Ethinyl Estradiol in human plasma.

A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF POMALIDOMIDE FROM HUMAN PLASMA

Objective: The present work aimed to develop a simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of pomalidomide and internal standard (ISTD) Fluconazole in human plasma.Methods: 50 µl of 0.1% formic acid was added to plasma samples prior to liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Xterra, RP18, 5 µ (50 x 4.6 mm) column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 12:88, v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of m/z 260.1 precursor ion to the m/z 148.8 for pomalidomide and m/z 307.1/238.0 for fluconazole.Results: The concentrations of nine working standards showed linearity between 9.998 to 1009.650 ng/ml (r2 ≥ 0.9968). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 53.86% for pomalidomide and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 1.32 to 4.03. The % stability of short term and long term stock solution stability studies was found to be 99.01% and 98.49% respectively.Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within limits. Thus the validated economical method was applied for pharmacokinetic studies of pomalidomide.

A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SOFOSBUVIR FROM HUMAN PLASMA

Objective: The present work aimed to develop a simple, rapid, specific and precise ultra-performance liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of sofosbuvir and internal standard (ISTD) Sofosbuvir-d3 in human plasma.Methods: Samples prepared by employing liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Gemini 5µ C18, 50 x 4.6 mm column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of 428.35/279.26 (m/z) for sofosbuvir and 431.38/282.37 (m/z) for sofosbuvir-d3.Results: The concentrations of ten working standards showed linearity between 4.063 to 8000.010ng/ml (r2 ≥ 0.9985). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 75.36% for sofosbuvir and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 0.35% and 3.09%. The % stability of short term and long term stock solution stability studies was found to be 97.25% and 98.81% respectively.Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within the acceptance limits. Thus the validated economical method was applied for pharmacokinetic studies of sofosbuvir.

Determination of betaine, a natural potential anti-inflammatory compound, using sildenafil as an internal standard in human plasma by high performance liquid chromatography–mass spectrometry

Betaine is a naturally occurring cationic functional group which shows a wide range of therapeutic activities. The present research work involves rapid; robust and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method developed and validated for s analysis of betaine in human plasma. This method involved a solid phase extraction and a subsequent analysis performed on an LC-MS system which contained the turbo spray ionization interface. Chromatographic separation was performed using a Gemini C18 column. Good linearity was observed over the concentration range of 10.00 ng/mL to 1203.26 ng/mL with a correlation coefficient (r² ≥ 0.9984). Sildenafil was used as the internal standard in the present method. The method involves simple isocratic chromatography conditions and mass spectrometric detection in the positive ionization mode. The intra-day and inter-day precision and accuracy of the quality control samples were within the acceptance criteria of ±15%. The total elution time was about 5 min which allows higher throughput. This validated method can be applied for analysis of real samples from a bioequivalence study involving administration of formulations containing betaine as their active therapeutic components.

A Validated UPLC-MS/MS Multi-mycotoxin Method for Nuts and Cereals for the Official Control in Cyprus within the EU Requirements

The validation of a rapid, reliable and sensitive method for the simultaneous determination of Aflatoxins (AFB1, AFB2, AFG1, AFG2), Ochratoxin A, Zearalenone, Deoxynivalenol, Fumonisins B1 and B2, T-2 and HT-2 toxins, in nuts (peanuts, pistachio and almonds) and cereals (maize and wheat) is reported. The method was developed and validated to fulfil the requirements of the official control of mycotoxins according to EU legislation. This method is based on a single extraction step using an acetonitrile/ water mixture followed by the analysis of the diluted crude extract using ultra high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The MS/MS detection was carried out using an electro spray-ionization interface (ESI) in positive ion mode. Matrix-matched calibration was used for the quantification of the mycotoxins, because of the absence of further clean up steps that reduce suppression/enhancement matrix effects. The method performance characteristics were determined after spiking blank samples on multiple levels. The mean recoveries of mycotoxins in spiked nuts ranged from 74.4% to 131.7%, while in cereals ranged from 52.8% to 113.9%. Relative standard deviations were lower than 20.4% for all target mycotoxins. Limits of detection and quantification for nuts and cereals ranged 0.08-30.0 and 0.25-99.0 μg/Κg, respectively.

Simultaneous determination of calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of Radix Astragali extraction for their pharmacokinetic studies by ultra-pressure liquid chromatography with tandem mass spectrometry.

A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC-MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters Oasis(TM) HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC™ BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r (2) > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-β-D-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-β-D-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.

A simple and rapid LC-MS/MS method for the determination of Enalapril in human plasma for pharmacokinetic and bioequivalence studies in korean healthy volunteers under fasting conditions

A simple and rapid liquid chromatography-tandem mass spectrometry method for Enalapril in human plasma was developed and applied to pharmacokinetic and bioequivalence test for 2 formulations of Enalapril (10 mg) capsules in healthy korean volunteers under fasting state. The analytes were extracted from plasma by simple protein precipitation by acetonitrile, separated on YMC C8 column using methanol-10 mM ammonium formate (80: 20, v/v) as the mobile phase, and detected by tandem mass spectrometry with Turbo IonSpray interface operating in the positive ion mode for Enalapril and Glibenclamide (IS) in MRM mode. The ionization was optimized using electro-spray ionization (ESI) (+) and selectivity was achieved by MS/MS analysis, m/z 376.447 → 234.1 for Enalapril and m/z 494.1 → 369.1 for IS. The assay exhibited good linearity in the concentration ranges of 1.0 ∼ 300 ng/mL for Enalapril in human plasma with lower limit of quantification (LLOQ) of 1.0 ng/mL. The chromatographic run time was approximately 2.0 min. No endogenous compounds were found to interfere with the analysis. The accuracy and precision were acceptable for concentrations over the standard ranges. The method was successfully applied to pharmacokinetic (PK) and bioequivalence (BE) studies by determination of Enalapril in the blood sample taken up to 12 h after oral administration of two Enalapril (10 mg) formulations and results from PK analysis suggested that the 2 types of 10 mg Enalapril tablets should be considered to be bioequivalent for both the extent and rate of absorption in normal volunteers.

Simultaneous targeted analysis of five active compounds in licorice by ultra-fast liquid chromatography coupled to hybrid linear-ion trap tandem mass spectrometry

An ultra-fast liquid chromatography with electrospray ionization tandem mass spectrometry (UFLC-ESI-MS/MS) method was developed for targeted analysis of 5 active compounds in licorice for the first time. The sample preparation procedure, chromatographic and mass spectrographic conditions were optimized. By using a Kinetex C18 100A column, the five compounds were separated within 8.0 min by gradient elution using methanol containing 0.1% acetic acid and 0.1% aqueous acetic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole/linear ion trap mass spectrometer equipped with a turbo ion spray interface in negative ionization mode (ESI(-)) and were simultaneously characterized and quantified based on the multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. All standard calibration curves expressed satisfactory linearity (r ≥ 0.9954) within a relatively wide range. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.15-4.56% and 0.81-3.95%, respectively. The recovery assays for the quantified compounds were between 97.33 and 100.4% with RSD values less than 4.27%. The proposed method was demonstrated to be simple, rapid, specific and reliable and was successfully applied for identification and quantification of 5 active compounds in 10 batches of licorice. The results showed that the contents of the 5 compounds in licorice from different sources were widely varied.

Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

A validated ultra-performance liquid chromatography mass spectrometric method (UPLC–MS/MS) was used for the simultaneous quantitation of candesartan (CN) and hydrochlorothiazide (HCT) in human plasma. The analysis was performed on UPLC–MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring (MRM) mode. The analytes were extracted using a liquid–liquid extraction (LLE) method by using 0.1mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00ng/mL whereas the upper limit of quantitation was 499.15ng/mL and 601.61ng/mL for CN and HCT respectively. CN d4 and HCT-13Cd2 were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0min run time using a C18 Phenomenex, Gemini NX (100 mm×4.6 mm, 5µm) column with organic mixture:buffer solution (80:20, v/v) at a flow rate of 0.800mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil (CNC) and HCT immediate release tablets with reference product in human subjects.

QUANTITATIVE ANALYSIS OF TEN DITERPENOIDS IN RAT BILE AFTER ORAL ADMINISTRATION OF Isodon rubescens EXTRACT BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY

Diterpenoids are the primary bioactive ingredients in Isodon rubescens named Dong Ling Cao in China which possess many pharmacological activities, including anti-tumor, anti-microbial, anti-inflammatory, anti-catastrophe, anti-oxidation, and anti-inflammatory effects. We employed a sensitive and selective high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) method for the quantification of ten diterpenoids in rat bile. The ten diterpenoids are effusanin A (1), lasiodonin (2), oridonin (3), epinodosin (4), nodosin (5), ponicidin (6), rabdoternin A (7), enmenol (8), lasiokaurin (9), and lasiokaurinol (10). The sulfamethoxazole was used as internal standard (IS). The bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). Chromatographic separation was performed on a C18 column with linear gradient elution using water and methanol at a flow rate of 0.8 mL/min. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode, respectively, using multiple–reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect, and several of stabilities have been validated for diterpenoids in rat bile samples. The results showed that this method is robust, specific, and sensitive and it can successfully fulfill the requirement of excretion study of the ten diterpenoids in Isodon rubescens.

SIMULTANEOUS DETERMINATION OF DIFFERENT CLASSES OF PLANT GROWTH REGULATOR IN HIGH WATER CONTENT AGRICULTURAL PRODUCTS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY AND TIME OF FLIGHT MASS SPECTROMETRY

Greenhouse farming is one of the most profitable forms of agriculture in Greece and in all areas of Europe. With the exception of pesticides that are widely used where residue levels are closely monitored either from the producers themselves or by the national authorities, the use of plant growth regulators (PGR) in plants grown in environmentally-controlled areas is also very common but not so closely controlled. In this study, a rapid and cost effective multiclass method is presented and validated for the simultaneous determination of various PGR, including 1-naphthylacetic acid, 2iP, 4-Chlorophenoxyacetic acid, 6-benzyladenine, forchlorfenuron, gibberellic acid, prohexadione, thidiazuron, 2.4-D, chlormequat, and mepiquat, in some of the most common greenhouse farming commodities (strawberries, as well as fruiting and brassica vegetables). The determination of the compounds was performed by two different mass spectrometry techniques, liquid chromatography-tandem mass spectrometry and time of flight mass spectrometry, both equipped with electron spray ionization interface. Supplemental materials are available for this article. Go to the publisher's online edition of the Journal of Liquid Chromatography to view the free supplemental file.

HPLC-ESI-MS/MS QUANTITATIVE METHOD FOR SIMULTANEOUS ANALYSIS OF FIVE BIOACTIVE CONSTITUENTS OF FORSYTHIA SUSPENSA IN RAT BILE AFTER ORAL ADMINISTRATION OF FORSYTHIA SUSPENSA EXTRACT

The compounds (+)-pinoresinol-β-D-glucoside (1), matai-resinol- 4′-O-glucoside (2), hyperin (3), phillyrin (4), and phillygenin (5) are the primary bioactive ingredients in Forsythia suspensa, which possesses many pharmacological activities, such as heat-clearing and detoxifying, free radical clearing, antioxidative, liver-protection, antibacterial, and so on. In the present study, we employed a sensitive and selective high-performance liquid chromatography electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of the five bioactive constituents of Forsythia suspensa in rat bile. Acetaminophen was used as the internal standard (IS). The bile samples were pretreated by liquid–liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C18 column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in negative mode using multiple-reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for the five bioactive constituents in rat bile samples. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five bioactive constituents after oral administration of Forsythia suspensa extract.

Liquid chromatography tandem mass spectrometry method for the simultaneous stereoselective determination of venlafaxine and its major metabolite, O‐desmethylvenlafaxine, in human plasma

A sensitive, accurate and highly stereoselective assay for the simultaneous determination of venlafaxine (VEN) and its equipotent metabolite, O-desmethyl venlafaxine (ODV), in human plasma was developed and validated. Analytes were simultaneously extracted from plasma using solid-phase extraction and detected by tandem mass spectrometry in positive ion mode with a turbo ion spray interface. Deuterium-labeled VEN and ODV were used as internal standards. Chromatographic separation was performed on a Chiral AGP column, using a time programmed gradient flow with a total run time of 16 min. The method has a lower limit of quantitation of 0.60 ng/mL. The assay was linear over a range 0.60-300.00 ng/mL for both the enantiomers of VEN and ODV, respectively, with coefficient of correlation > 0.99. The extraction recoveries were >77.0% on an average for all the four analytes. The analytes were found stable in plasma through three freeze (-15 °C) and thaw cycles and under storage at room temperature for 8 h, and also in mobile phase at 10 °C for 54 h. The method has shown good reproducibility, with intra- and inter-day variation coefficients < 9%, for all the analytes, and has proved to be very reliable for analysis of VEN and its metabolite in clinical study samples.

Sensitive quantification of roflumilast and roflumilast N-oxide in human plasma by LC–MS/MS employing parallel chromatography and electrospray ionisation

A high throughput bioanalytical method based on semi-automated liquid extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the sensitive quantification of roflumilast and its metabolite roflumilast N-oxide, a phosphodiesterase (PDE) inhibitor in human plasma and serum. The sample work-up procedure comprised liquid extraction using penta-deuterated analogues of both analytes as internal standards. Chromatography was performed on C18 revered phase analytical columns at a flow rate of 0.5mL/min in the dual column mode employing a column switching technique and a linear gradient from 18% to 54% acetonitrile in 0.005M aqueous ammonium acetate containing 0.006% formic acid. Mass spectrometry was performed on an API 4000 instrument in the positive ion SRM-mode (selected reaction monitoring) with the Turbo-V® ionspray interface. The method showed linear detector responses over the entire calibration range between 0.1ng/mL (lower limit of quantification (LLOQ)) and 50ng/mL (upper limit of quantification (ULOQ)) for both analytes. Linear regression analysis with concentration-squared weighting (1/x2 for roflumilast and 1/x for roflumilast N-oxide) yielded inaccuracy and precision values <15% and coefficients of correlation (r) for the calibration curves >0.99 for both analytes.

Development and validation of LC–MS/MS assays for the quantification of E7080 and metabolites in various human biological matrices

To support clinical pharmacokinetic studies with the anticancer agent E7080 (lenvatinib), liquid chromatography tandem mass spectrometry (LC–MS/MS) methods were developed for the quantification of E7080 and four of its metabolites in human plasma, urine and faeces and of E7080 in whole blood. Cross-analyte interferences between metabolites and parent compound were expected and therefore accounted for early in the method development. Plasma, urine and faeces samples were extracted with acetonitrile. Chromatographic separation was achieved on a 50mm×2.1mm I.D. XTerra MS C18 column, with a 0.2mL/min flow and gradient elution starting with 100% formic acid in water, followed by an increasing percentage of acetonitrile. Whole blood samples were extracted with diethyl ether and extracts were injected on a 150mm×2.1mm I.D. Symmetry Shield RP8 column. Detection was performed using an API3000 triple quadrupole mass spectrometer, with a turbo ion spray interface, operating in positive ion mode. Using 250μL of plasma, E7080 and its metabolites could be quantified between 0.25 and 50.0ng/mL. The quantifiable ranges of E7080 in whole blood, urine and faeces were 0.25–500ng/mL, 1.00–500ng/mL and 0.1–25μg/g, using sample volumes of 250μL, 200μL and 250mg, respectively. Calibration curves in all matrices were linear with a correlation coefficient (r2) of 0.994 or better. At the lower limit of quantification, accuracies were within ±20% of the nominal concentration with CV values less than 20%. At the other concentrations the accuracies were within ±15% of the nominal concentration with CV values below 15%. The developed methods have successfully been applied in a mass balance study of E7080.