LC–MS–MS Determination of Rotenone, Deguelin, and Rotenolone in Human Serum

For the first time we report a rapid and sensitive LC–MS–MS method for quantification of rotenone, deguelin, and rotenolone in human serum. The analytical procedure involves extraction with ethyl acetate without further clean-up. The active ingredients were separated on a C₈ reversed-phase column by isocratic elution. Eleven simultaneous transitions of precursor ions were monitored. Excellent selectivity and sensitivity enables quantification and identification of low levels of rotenoids (LOD 2 ng mL^?1, LOQ 5 ng mL^?1) in human serum.     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Ion-Pair Single-Drop Microextraction Determinations of Degradation Products of Chemical Warfare Agents in Water

Ion-pair single-drop microextraction (SDME) coupled to gas chromatography–mass spectroscopy (GC–MS) methods for the determination of four degradation products of chemical warfare agents were investigated in water. Acidic analytes were converted into their ion-pair complexes with cation surfactants in aqueous sample and then extracted into the organic single drop containing the derivatising agent. Upon injection, the analytes were derivatised in the GC injection hot port. Parameters, such as type of extraction solvent, ion-pairing (IP) reagent, reagent concentration, salt concentration, stirring speed and pH, were all optimized. This method is reproducible for spiked water sample for four different analytes (RSDs < 9.33 %, n = 5) and linear (r ² > 0.9945). The limit of detection (LOD) is in the range of 0.08–0.01 ng mL⁻¹ (S/N = 5) under GC–MS selected ion monitoring mode. The method was successfully applied to the proficiency test samples from the Organization for Prohibition of Chemical Weapons (OPCW).

     

Simultaneous LC–MS–MS Analysis of Danshensu, Salvianolic Acid B, and Hydroxysafflor Yellow A in Beagle Dog Plasma, and Application of the Method to a Pharmacokinetic Study of Danhong Lyophilized Powder for Injection

A reliable and sensitive liquid chromatographic–tandem mass spectrometric method, with rutin as internal standard, has been developed and validated for simultaneous determination of danshensu, salvianolic acid B (SAB), and hydroxysafflor yellow A (HSYA) in beagle dog plasma. Plasma samples spiked with the analytes were extracted by solid-phase extraction and the analytes were separated on a 250 × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–acetonitrile–0.5% formic acid 20:25:55 (v/v) as mobile phase at a flow rate of 1 mL min^?1. LC–MS–MS analysis was performed with a Finnigan TSQ triple-quadrupole tandem mass spectrometer operated in negative-ion selected-reaction-monitoring mode, using electrospray ionization. The accuracy and precision of the method were acceptable and linearity was good over the range 20–4,000 ng mL^?1 for danshensu, 50–10,000 ng mL^?1 for SAB, and 10–2,000 ng mL^?1 for HSYA. The method was successfully applied to a pharmacokinetic study of a traditional Chinese medicinal preparation, Danhong lyophilized powder for injection.     

LC–MS–MS Method for Simultaneous Analysis of Abacavir and Lamivudine in Human Plasma, and Its Application to a Bioequivalence Study

A rapid and sensitive LC–MS–MS method has been developed and validated for simultaneous analysis of abacavir (ABA) and lamivudine (LAM) in human plasma. The analytes were extracted from human plasma by SPE. Nelfinavir (NEL) and emtricitabine (EMT) were used as the internal standards for ABA and LAM, respectively. An RP18 column enabled chromatographic separation of the analytes. The method involves simple isocratic chromatography and MS detection in positive-ionization mode. Validation of the method showed response was a linear function of concentration in the ranges 100.0–7000.0 ng mL^?1 for ABA and 80.0–5000.0 ng mL^?1 for LAM. At the LOQ levels, inter-run and intra-run precision were within 5.80 and 3.51%, respectively, for ABA and within 4.68 and 3.16%, respectively, for LAM. Overall recovery for ABA and LAM was 59.32 and 105.18%, respectively. Total elution time was 2 min only, which enabled quantification of more than 200 plasma samples per day. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.     

LC–MS–MS Method to Simultaneously Determine Six Probe Drugs for CYP450 Isozymes in Human Liver Microsomes

Here, we report a rapid and specific method based on high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) capable of quantifying six CYP450-specific probe substrates in human liver microsomal incubation mixtures simultaneously. These analytes were prepared by single-step extraction and detected in one run by switching polarity of electrospray ionization mode three times. Following optimization of the chromatographic conditions, the peaks were well separated, and retention times ranged between 2.0 and 8.4 min. The total run time for a single injection was within 9 min. This method was fully validated over linear range of 18.8–3,000.0 ng mL^?1 for diclofenac, 0.8–3,000.0 ng mL^?1 for dapson, 1.5–3,000.0 ng mL^?1 for dextromethorphan, 2.0–4,000.0 ng mL^?1 for omeprazole, 75.0–3,000.0 ng mL^?1 for chlorzoxazone and 0.8–3,000.0 ng mL^?1 for phenacetin using diazepam as internal standard. Samples were prepared by protein precipitation and analyzed on the LC–MS–MS equipped with ESI interface. For each analyte, inter- and intra-day precision (RSD%) were <15 % and accuracy was within 85–115 %. The specificity, precision, accuracy, stabilities and matrix effect were evaluated.     

LC–MS/MS Determination of Antihypertension Drugs in Rat Plasma and Urine: Applications to Pharmacokinetics

A sensitive, rapid and reproducible LC–MS/MS method for the determination of olmesartan (OLM), amlodipine (ALM) and hydrochlorothiazide (HCZ) in rat plasma and urine has been developed and validated. Irbesartan (IRB) was used as an internal standard. The analytes were separated on a Waters XTerra-C₁₈ column using gradient elution with acetonitrile and 10 mM ammonium acetate buffer (pH 3.5, adjusted with acetic acid) at a flow rate of 1.0 mL min^?1. The three analytes were ionized by positive ion electrospray using multiple-reaction monitoring (MRM) mode to monitor precursor?→?product ion transitions m/z 447.31?→?234.97 for OLM, 408.87?→?238.18 for AML and 290.1?→?204.85 for HCZ. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range 0.4–100 ng mL^?1 for AML, 0.2–100 ng mL^?1 for OLM, 0.1–100 ng mL^?1 for HCZ. The mean concentrations (C_max) are 10.32, 587, and 3.4 for OLM, ALM, and HCZ, respectively, by the oral administration of 15 mg kg^?1 of each analyte.     

Determination of Tangeretin in Rat Plasma by LC-Electrospray-Ion Trap MS

A selective, sensitive, and accurate method has been developed and validated for the quantification of tangeretin in rat plasma. The application of LC-electrospray-ion trap mass spectrometry in full scan and multiple reactions monitoring modes were investigated. Following solid phase extraction using a hydrophilic–lipophilic balance cartridge, the analytes were separated on a C₁₈ column using an isocratic mobile phase composed of acetonitrile/water (50:50, v/v) containing 0.3% formic acid. In full scan mode, the LOQ was 2 ng mL^?1. The standard calibration curve was linear (R ² = 0.9999) over the concentration range 2–200 ng mL^?1. The precision over the concentration range was within 15% (RSD) and the accuracy was ranged from 86 to 115%. In multiple reaction monitoring mode, the LOQ was 1 ng mL^?1 and the standard calibration curve was linear (R ² = 0.9976) over the concentration range 1–100 ng mL^?1 with a precision of 12% and accuracy rangeing from 91 to 113%.     

GC–MS Determination of Atenolol Plasma Concentration after Derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide

An analytical procedure was developed and validated for the determination of atenolol in human plasma. Atenolol and metoprolol (internal standard) were extracted from human plasma with a mixture of chloroform and butanol at basic pH. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed by GC–MS. Calibration curves were linear over the concentration range 15–250 ng mL^?1. Intra- and inter-day precision values for atenolol in human plasma were less than 7.4, and accuracy (relative error) was better than 6.4%. Recovery of atenolol from human plasma averaged 90.46%. The limits of detection (LOD) and quantitation (LOQ) of atenolol were 5.0 and 15 ng mL^?1. This method was successfully applied to six patients with hypertension who had been given an oral tablet of 50 mg atenolol.     

Ion-Pair Single-Drop Microextraction Determinations of Degradation Products of Chemical Warfare Agents in Water

Ion-pair single-drop microextraction (SDME) coupled to gas chromatography–mass spectroscopy (GC–MS) methods for the determination of four degradation products of chemical warfare agents were investigated in water. Acidic analytes were converted into their ion-pair complexes with cation surfactants in aqueous sample and then extracted into the organic single drop containing the derivatising agent. Upon injection, the analytes were derivatised in the GC injection hot port. Parameters, such as type of extraction solvent, ion-pairing (IP) reagent, reagent concentration, salt concentration, stirring speed and pH, were all optimized. This method is reproducible for spiked water sample for four different analytes (RSDs < 9.33 %, n = 5) and linear (r ² > 0.9945). The limit of detection (LOD) is in the range of 0.08–0.01 ng mL^?1 (S/N = 5) under GC–MS selected ion monitoring mode. The method was successfully applied to the proficiency test samples from the Organization for Prohibition of Chemical Weapons (OPCW).     

Determination of Pantoprazole, Rabeprazole, Esomeprazole, Domperidone and Itopride in Pharmaceutical Products by Reversed Phase Liquid Chromatography Using Single Mobile Phase

A simple, sensitive, and precise high performance liquid chromatographic method for the analysis of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride, with ultraviolet detection at 210 nm, has been developed, validated, and used for the determination of compounds in commercial pharmaceutical products. The compounds were well separated on a Hypersil BDS C18 reversed-phase column by use of a mobile phase consisting of 0.05 M, 4.70 pH, potassium dihydrogen phosphate buffer - acetonitrile (720:280 v/v) at a flow rate of 1.0 mL min^?1. The linearity ranges were 400–4,000 ng mL^?1 for pantoprazole, 200–2,000 ng mL^?1 for rabeprazole, 400–4,000 ng mL^?1 for esomeprazole, 300–3,000 ng mL^?1 for domperidone and 500–5,000 ng mL^?1 for itopride. Limits of detection (LOD) obtained were: pantoprazole 147.51 ng mL^?1, rabeprazole 65.65 ng mL^?1, esomeprazole 131.27 ng mL^?1, domperidone 98.33 ng mL^?1 and itopride 162.35 ng mL^?1. The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of pantoprazole, rabeprazole, esomeprazole, domperidone and itopride using single mobile phase.     

Determination of Milnacipran in Human Plasma Using GC–MS

A new, simple, and fully validated gas chromatography–mass spectrometry (GC–MS) method was presented for quantitative analysis of milnacipran (MNP) in human plasma. MNP was efficiently derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before analysis. The role of catalyst, temperature, time, solvent on the trimethylsilylation reaction were evaluated. The proposed method was fully validated by assessment of the following parameters: limits of detection and quantitation, precision, accuracy, linearity, specificity, stability, extraction recovery and robustness/ruggedness. The limit of quantitation (LOQ) was 30 ng mL^?1. The calibration curve was linear (r ² > 0.9988) in the range 30–500 ng mL^?1. The method was found specific, precise, accurate, selective and reliable according to validation data. This developed method was successfully applied to determine the steady state concentration of MNP in patients.     

An Experimental Design Approach to Selecting the Optimum LC Conditions for the Determination of Local Anaesthetics

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anaesthetics: lidocaine, proparacaine, bupivacaine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C₈ (4.6 × 2.5 mm i.d., particle size 5 μm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 mL min^?1. Local anaesthetics detection was performed by UV-Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacaine and oxybuprocaine were 5.74, 9.28, 16.84 and 26.26 min, respectively. HPLC-UV-Vis method was linear in the range of 50–5,000 ng mL^?1 for lidocaine and proparacaine and 100–5,000 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of detection (LOD) was 25 ng mL^?1 for lidocaine, proparacaine and 30 ng mL^?1 for bupivacaine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng mL^?1 for lidocaine, proparacaine and 100 ng mL^?1 for bupivacaine, oxybuprocaine. In intra-day and inter-day precision and accuracy analysis, the relative standard deviation was found to be less than 8%.     

Determination of Protoberberine Alkaloids in Coptis chinensis by Microextraction and High Performance Liquid Chromatography

Sample preparation technique based on an organic filter membrane (pH-resolved filter membrane microextraction) (pH-RFMME) was developed, coupled with high-performance liquid chromatography, and used to determine protoberberine alkaloids (jatrorrhizine, epiberberine, coptisine, palmatine, and berberine) in Coptis chinensis at different pH values through a one-step procedure. This green procedure provides a desirable sample pretreatment technology. The main variables affecting the extraction such as filter membrane area (or volumes of extraction solvents), sample pH, eluent pH, ionic strength, extraction stirring rate, extraction time, and sample volume were optimized. Under the optimized conditions, the enrichment factors of the analytes were 40.4–52.0, the linear ranges were 3.2–6250 ng · mL^?1 for jatrorrhizine and epiberberine, 6.0–12000 ng · mL^?1 for coptisine, 1.8–3600 ng · mL^?1 for palmatine, and 18.8–18800 ng · mL^?1 for berberine, with r ² ≥ 0.9945. The limits of detection were less than 0.3 ng · mL^?1. Satisfactory recoveries (84.8%–115.5%) and precision (1.8%–10.0%) were also achieved. These results confirmed that pH-RFMME is a simple, rapid, practical, and environmentally friendly method to isolate analytes that exhibit significant differences in acidity or alkalinity from complex samples.     

Simultaneous Estimation of Atorvastatin Calcium, Ramipril and Aspirin in Capsule Dosage Form by RP-LC

A simple, sensitive, precise and accurate reversed phase liquid chromatographic method has been developed for the simultaneous estimation of atorvastatin (AT) calcium, ramipril (RA) and aspirin (AS) from capsule dosage form. The method was developed using a Phenomenex Luna C₁₈ (250 mm, 4.6 mm i.d., 5 µm) column with a mobile phase consisting of 0.1%, orthophosphoric acid buffer:acetonitrile:methanol (45:50:5 v/v/v), pH 3.3, at a flow rate of 1 mL min^?1. Detection was carried out with ultra-violet detection at 210 nm. The retention times were about 12.19, 2.35, and 3.95 min for AT calcium, RA and AS, respectively. The developed method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linearity ranges were 1–6 µg mL^?1 for AT calcium, 0.5–3 µg mL^?1 for RA and 7.5–45 µg mL^?1 for AS with mean recoveries of 100.59 ± 0.68, 100.62 ± 0.83 and 100.49 ± 0.73% for AT calcium, RA and AS, respectively. Limit of detection obtained were 29.85 ng mL^?1 for AT calcium, 4.71 ng mL^?1 for RA and 85.13 ng mL^?1 for AS. Impurity of salicylic acid was found in capsule dosage form at the retention time of about 4.84 min. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

GC Determination of Prilocaine HCl in Human Plasma: Analytical Application to Real Samples

A gas chromatographic (GC) method with a rapid and simple sample preparation was developed and validated for determination of prilocaine in human plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC method was 50–300 ng mL^?1 in plasma. Intra- and inter-day precision, expressed as the relative standard deviation (RSD) were less than 4.5%, and accuracy (relative error) was better than 8.0% (n = 6). The analytical recovery of prilocaine HCl from plasma has averaged 96.5% and the recovery of internal standard (lidocaine HCl) reached 96.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method for plasma were 50 and 40 ng mL^?1, respectively. Also the developed and validated method was applied to three healthy volunteers to whom a local anaesthesia with citanest was administered.     

LC-DAD Determination of Fleroxacin in Bulk and Pharmaceutical Dosage Forms

Fleroxacin is a third generation fluoroquinolone with broad spectrum antibacterial activity. In this work an LC-DAD method for the analysis of fleroxacin was developed and validated using UV detection at 286 nm. The method was validated for linearity, precision, robustness, LOD, LOQ, specificity and accuracy at concentrations of 0.2–20.0 μg mL⁻¹ and r ² = 1. The LOD and LOQ were 0.059 and 0.197 μg, respectively, the recoveries were 99.92–102.0% and the CV was less than 2.0%. The LC-DAD validated method provided analytical sensitivity, specificity and reproducibility suitable for quality control analysis.

     

Magnetic Nanoparticle-Based Micro-Solid Phase Extraction and GC�CMS Determination of Oxadiargyl in Aqueous Samples

A new facile, rapid, inexpensive, and sensitive method based on magnetic micro-solid phase extraction (M-??-SPE) coupled to gas chromatography?Cmass spectrometry (GC?CMS) was developed for determination of the herbicide oxadiargyl in environmental water samples. The feasibility of employing non-modified magnetic nanoparticles (MNPs) as sorbent was examined and applied to perform the extraction process. Influential parameters affecting the extraction efficiency along with desorption conditions were investigated and optimized. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) of the method under optimized conditions were 0.005 and 0.030 ng mL^?1, respectively. The relative standard deviations (RSD) (n = 3) at a concentration of 0.10 ng mL^?1 was 11%. The calibration curve of oxadiargyl showed linearity in the range of 0.050?C0.50 ng mL^?1. The developed method was successfully applied to the extraction of oxadiargyl from spiked tap water and Zayande-Rood River water samples and the relative recoveries of 98 and 94% were obtained, respectively.     

Development and Validation of an HPLC–UV–Vis Method for the Determination of Proparacaine in Human Aqueous Humour

A sensitive high performance liquid chromatographic method has been developed and validated for the determination of proparacaine in human aqueous humour. The procedure involved extraction of proparacaine from aqueous humour with cyclohexane. The separation was achieved using a Bondesil C₈ (250 × 4.6 mm i.d., particle size 5 μm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH 3.0, 20 mM) (30:70, v/v). Proparacaine and lidocaine (internal standard, IS) detection was performed by UV–Vis detector at 220 nm. The retention times for proparacaine and IS were 12.01 and 5.58 min, respectively. HPLC–UV–Vis method was linear in the range of 75–4,000 ng mL⁻¹. The limit of detection (LOD) was 25 ng mL⁻¹ and the limit of quantification (LOQ) of proparacaine was found to be 75 ng mL⁻¹ (RSD ≤ 15%, n = 6). In intra- and inter-day precision and accuracy analysis, the relative standard deviation was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Recovery of proparacaine from human aqueous humour was 99.98% at 500 ng mL⁻¹. Proparacaine solutions were stable at least 6 months at +4 and −20 °C. Proparacaine levels of aqueous humour in fifteen volunteers’ were in the range of 80.21 and 459.00 ng mL⁻¹. According to system suitability tests and Shewhart’s quality control charts the proparacaine responses were in the acceptance ranges. Developed method was providing a sufficient quality at least over 3 months for determination of proparacaine in human aqueous humour.

     

Simultaneous determination of five triterpenic acids in four Corydalis herb medicines by reversed-phase high performance liquid chromatography–fluorescence-mass spectrometer (RP-HPLC–FLD-MS) based on pre-column derivatization

A rapid and sensitive reversed-phase high performance liquid chromatography– fluorescence-mass spectrometer (RP-HPLC–FLD-MS) method based on pre-column derivatization using 2-(5-benzoacridine)ethyl-p-toluenesulfonate (BAETS) as labelling reagent has been developed for simultaneous determination of five triterpenic acids (asiatic acid (AA), maslinic acid (MA), corosolic acid (CA), oleanolic acid (OA), and betulinic acid (BA). The presented method was validated for linearity (correlation coefficient R² > 0.9994), precision (RSD 2.1%–3.9%) and reproducibility (RSD 0.01%–2.1%). The limits of detection (LODs) and the limits of quantitation LOQs were within the range of 0.71–1.02 ng mL^?1 and 2.28–3.24 ng mL^?1, respectively. The proposed method was successfully applied to simultaneously determine five triterpenic acids of four Corydalis plants and showed satisfactory reproducibility and credibility. Moreover, several main parameters affecting extraction procedure and derivatization efficiency were investigated by response surface methodology (RSM), respectively. Triterpenic acid content in four Corydalis plants was measured according to the established method and the results indicated that triterpenic acid contents were various in different organs and herbs.