Micellar liquid chromatographic method for the determination of ephedrine and pseudoephedrine in human serum by direct inject of the sample with simple dilution

A micellar high-performance liquid chromatographic method was developed to simultaneously determine ephedrine and pseudoephedrine in human serum. The serum sample pretreatment was a simple dilution in a micellar solution, filtration, and direct injection, thus avoiding time-consuming and tedious steps. Hence, there is no need to use an internal standard. The serum samples were analyzed using a mobile phase containing 1.50?×?10^?1?mol/L sodium dodecyl sulfate and 0.02?mol/L sodium dihydrogen phosphate with 7.5% (v/v) 1-propanol at pH 3.0, running at 1.0?mL/min by an Inertsil C18 (150?×?4.6?mm, 5?µm) column at 30°C. The UV wavelength was set at 210?nm. The developed method was validated by linearity (r?>?0.9990) and intra- and inter-day precisions of ephedrine and pseudoephedrine (relative standard deviation; RSD%, 0.04–10.40, and RSD %, 0.30–10.25, respectively), LODs for ephedrine and for pseudoephedrine was 2.63 and 2.70?µg/mL, respectively; lower limit of quantification for ephedrine and for pseudoephedrine was 4.38 and 4.51?µg/mL, respectively. Finally, the proposed method was applied to investigate ephedrine and pseudoephedrine in real human serum samples after oral administration of Kechuanning Koufye including Ephedra herb. It is environmentally friendly, easy-to-handle, and feasible method for routine analysis in clinical laboratory.     

Development and Validation of High-Throughput Bioanalytical Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Newly Synthesized Antitumor Carbonic Anhydrase Inhibitors in Human Plasma

In the present study, a sensitive and fully validated bioanalytical high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitative determination of three newly synthesized carbonic anhydrases inhibitors (CAIs) with potential antitumor activity in human plasma. The analytes and the internal standard (IS) were extracted using 1.5 mL acetonitrile from only 450 µL aliquots of human plasma to achieve the desired protein precipitation. Chromatographic separations were achieved on Phenomenex Kinetex^® C₁₈ column (100 × 4.6 mm, 2.6 µm) using a binary gradient elution mode with a run time of less than 6 min. The mobile phase consisted of solvent (A): 0.1% formic acid in 50% methanol and solvent B: 0.1% formic acid in acetonitrile (30:70, v/v), pumped at a flow rate of 0.8 mL/min. Detection was employed using triple quadrupole tandem mass spectrometer (API 3500) equipped with an electrospray ionization (ESI) source in the positive ion mode. Multiple reaction monitoring (MRM) mode was selected for quantitation through monitoring the precursor-to-parent ion transition at m/z 291.9 → 173.0, m/z 396.9 → 225.1, m/z 388.9 → 217.0, and m/z 146.9 → 91.0 for AW-9a, WES-1, WES-2, and Coumarin (IS), respectively. Linearity was computed using the weighted least-squares linear regression method (1/x²) over a concentration range of 1–1000, 2.5–800, and 5–500 ng/mL for AW-9a, WES-1, and WES-2; respectively. The bioanalytical LC-MS/MS method was fully validated as per U.S. Food and Drug Administration (FDA) guidelines with all respect to linearity, accuracy, precision, carry-over, selectivity, dilution integrity, and stability. The proposed LC-MS/MS method was applied successfully for the determination of all investigated drugs in spiked human plasma with no significant matrix effect, which is a crucial cornerstone in further therapeutic drug monitoring of newly developed therapeutic agents.     

δ13C, δ15N and δ2H isotope ratio mass spectrometry of ephedrine and pseudoephedrine: application to methylamphetamine profiling

Conventional chemical profiling of methylamphetamine has been used for many years to determine the synthetic route employed and where possible to identify the precursor chemicals used. In this study stable isotope ratio analysis was investigated as a means of determining the origin of the methylamphetamine precursors, ephedrine and pseudoephedrine. Ephedrine and pseudoephedrine may be prepared industrially by several routes. Results are presented for the stable isotope ratios of carbon (δ¹³C), nitrogen (δ¹⁵N) and hydrogen (δ²H) measured in methylamphetamine samples synthesized from ephedrine and pseudoephedrine of known provenance. It is clear from the results that measurement of the δ¹³C, δ¹⁵N and δ²H stable isotope ratios by elemental analyzer/thermal conversion isotope ratio mass spectrometry (EA/TC‐IRMS) in high‐purity methylamphetamine samples will allow determination of the synthetic source of the ephedrine or pseudoephedrine precursor as being either of a natural, semi‐synthetic, or fully synthetic origin. Copyright © 2009 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.     

Differential pharmacokinetics and the brain distribution of morphine and ephedrine constitutional isomers in rats after oral administration with Keke capsule using rapid‐resolution LC–MS/MS

Opioid and ephedra alkaloids known as the active ingredients for Keke capsule, which is used to treat coughs and bronchial asthma, could have potential adverse effects on the central nervous system. Therefore, an efficient, sensitive rapid‐resolution LC–MS/MS method for the simultaneous determination of morphine, ephedrine, and pseudoephedrine in rat plasma and brain tissue homogenate has been developed. The method was validated in the plasma and brain tissue samples, showed good linearity over a wide concentration range (r² > 0.99). The intra‐ and interday assay variability was less than 15% for all analytes, and the accuracy was between ?8.8 and 5.7%. The study provided the pharmacokinetics profiles and the brain regional distribution of the three active alkaloids after oral administration of Keke capsule. The results also indicated that significant difference in pharmacokinetics parameters of the epimers was observed between ephedrine and pseudoephedrine.     

High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15 mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R² > 0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87–110.99%), limits of detection (5.35–5.76 µg/mL), and limits of quantification (20 µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents of three batches of Ephedrae Herba from three pharmaceutical companies.     

Determination of diastereoisomeric alkaloids in urine by capillary electrophoresis with electrochemiluminescence detection

A new and simple capillary electrophoresis with electrochemiluminescence detection was developed for the separation and the quantification of a pair of diastereoisomenc alkaloids（ephedrine and pseudoephedrine）.The limits of detection（S/N = 3） were 4.5×10^-8 mol/L for ephedrine and 5.2×10^-8 mol/L for pseudoephedrine,respectively.The RSDs of migration time and peak area were less than 1.3 and 2.5%（n = 5）,respectively.The applicability of the propose method was illustrated in the determination of ephedrine and pseudoephedrine in human urine,ephedrine in nasal drops,and the monitoring of pharmacokinetics for pseudoephedrine.     

HPLC Determination of Imidazoles with Variant Anti-Infective Activity in Their Dosage Forms and Human Plasma

A suitable HPLC method has been selected and validated for rapid simultaneous separation and determination of four imidazole anti-infective drugs, secnidazole, omeprazole, albendazole, and fenbendazole, in their final dosage forms, in addition to human plasma within 5 min. The method suitability was derived from the superiority of using the environmentally benign solvent, methanol over acetonitrile as a mobile phase component in respect of safety issues and migration times. Separation of the four anti-infective drugs was performed on a Thermo Scientific^® BDS Hypersil C₈ column (5 µm, 2.50 × 4.60 mm) using a mobile phase consist of MeOH: 0.025 M KH₂PO₄ (70:30, v/v) adjusted to pH 3.20 with ortho-phosphoric acid at room temperature. The flow rate was 1.00 mL/min and maximum absorption was measured with UV detector set at 300 nm. Limits of detection were reported to be 0.41, 0.13, 0.18, and 0.15 µg/mL for secnidazole, omeprazole, albendazole, and fenbendazole, respectively, showing a high degree of the method sensitivity. The method of analysis was validated according to Food and Drug Administration (FDA)guidelines for the determination of the drugs, either in their dosage forms with highly precise recoveries, or clinically in human plasma, especially regarding pharmacokinetic and bioequivalence studies.     

Quick bioanalytical method of quantification and validation of sulbactam in plasma using high performance liquid chromatography

A quick method of quantitative determination of sulbactam in human plasma, using liquid chromatography-UV spectroscopy, has been developed and validated. After derivatization with imidazole, plasma samples were treated by direct deproteinization with acetonitrile as an extraction solvent. After ultracentrifugation, sulbactam extract was directly injected onto the LC column. Chromatographic separation was performed on TSK Gel Super ODS (50 mm × 4.6 mm i.d., 2 μm) using methanol and phosphate buffer with tetrabutylammonium hydroxide solution as a mobile phase. Gradient elution was employed. The method was fully validated according to the United States Food and Drug Administration requirements (linearity, precision, trueness, quantification limit, detection limit, recovery, specificity and stability). The calibration curves were linear within the concentration range of 0.05–4.0 μg mL⁻¹. Good method/system precision and accuracy of the method were demonstrated.      

Development of CE-C4D Method for Determination Tropane Alkaloids

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C⁴D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) β-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters—precision (CV): 1.07–2.08%, accuracy of the assay (recovery, RE): 101.0–102.7% and matrix effect (ME): 92.99–94.23%. Moreover, the optimized CE-C⁴D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.     

Method for derivatization of ephedrine and pseudoephedrine in nonaqueous media and determination by nonaqueous capillary electrophoresis with laser induced fluorescence detection

A nonaqueous capillary electrophoresis with laser-induced fluorescence detection method was developed for the quantification of ephedrine and pseudoephedrine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol in nonaqueous media. The derivatization was made in off-line mode. By a series of optimizations, a derivatization buffer composed of 40 mm ammonium acetate and 20% acetonitrile and a running buffer composed of 80 mm ammonium acetate and 3% acetic acid were applied for the derivatization and separation of ephedrine and pseudoephedrine, respectively. Linear relationships for ephedrine and pseudoephedrine were obtained in the range 1.23-19.60 mg/L (correlation coefficients 0.9970 for ephedrine and 0.9994 for pseudoephedrine), and the detection limits for ephedrine and pseudoephedrine were 0.014 and 0.011 mg/L, respectively. The method was applied to the analysis of ephedrine and pseudoephedrine in four preparations with recoveries in the range 93.9-105.1%.     

Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a Diamonsil^TM C₁₈ column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R² ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.     

In vitro/in vivo screening of oxidative homeostasis and damage to DNA,protein, and lipids using UPLC/MS-MS

Multiple analytical methods are required to comprehensively assess oxidative homeostasis and specific damage to macromolecules. Our aim was to develop a straightforward strategy for the fast assessment of global oxidative status and specific damage to DNA, proteins, and lipids. To this end, an analytical method, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS), was developed and validated for the quantification of 16 oxidative stress (OS) biomarkers. Some of these markers were unstable; thus, an easy sample treatment procedure, including fractionation and derivatization, was set up. The method was validated according to Food and Drug Administration (FDA) guidelines, and it provided good results in terms of intra- and inter-day precision (≤17.2 and 16 %, respectively), accuracy (relative error measurement between ?16.6 and 19.8 %), and linearity (R ²?>?0.994). The approach was applied to determine the oxidative insult provoked to cultured rat hepatocytes by cumene hydroperoxide and to analyze the liver and serum samples from patients diagnosed with nonalcoholic steatohepatitis. In both studies, significant differences were found if compared to the corresponding control groups; interestingly, ophthalmic acid was shown as an OS biomarker in both models for the first time. A key advantage of the novel approach in comparison with former multi-method approaches is that now a single method is applied to assess the 16 OS biomarkers. Its comprehensive capacity to profile oxidative homeostasis and damage in both in vitro and clinical samples has been illustrated, which indicates that the proposed approach is a good choice to evaluate whether OS is involved in physiological signals, diseases, or toxic events and to what extent.     

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

基于中空纤维液相微萃取的麻黄碱和伪麻黄碱优势构象的确定及含量测定

利用中空纤维液相微萃取方法(HF-LPME)分析麻黄碱和伪麻黄碱在不同基质中的优势构象,阐明了麻黄碱和伪麻黄碱的萃取机理;结合高效液相色谱(HPLC)建立了微量麻黄碱和伪麻黄碱的分离测定方法。以聚偏氟乙烯中空纤维为有机溶剂载体,正己醇为萃取溶剂,麻黄碱和伪麻黄碱的NaOH(5 mol/L)溶液为样品相,0.01 mol/L H2SO4溶液为接收相,在1200 r/min转速下萃取35 min,收集萃取液直接进行HPLC分析。麻黄碱和伪麻黄碱在水溶液中的线性范围为5～100 μg/L,检出限分别为1.9 μg/L和1.2 μg/L,富集倍数分别为38和61倍,平均回收率分别为100.6%±1.2%和103.2%±3.5%;在鼠尿液中的线性范围为100～5×104 μg/L,检出限分别为30 μg/L和42 μg/L,富集倍数分别为20和17倍,平均回收率分别为108.4%±4.4%和106.1%±5.4%。研究表明该方法操作简单,选择性高,适用于微量麻黄碱的含量测定和分析。     

LC–MS–MS Assay for Simultaneous Quantification of Fexofenadine and Pseudoephedrine in Human Plasma

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL⁻¹ (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.     

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d‐methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d‐methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C₁₈ MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH₂PO₄–acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d‐methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high‐purity d‐methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high‐purity methamphetamine profiling. Copyright © 2011 John Wiley & Sons, Ltd.     

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3′-Monoxime in Plasma and Cell Culture Medium

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3′-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole) and KD85 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.     

Individual and simultaneous determination of ephedrine and phenylephrine by use of kinetic methodology

Kinetic methodology was for the first time used to develop a simple photometric method for the determination for ephedrine and phenylephrine using the stopped-flow mixing technique. The method involves oxidation of these compounds and kinetic monitoring of the reactions of the aldehydes formed with 2-methylbenzothiazoline-2-one hydrazone in the presence of iron (III) chloride to give the corresponding coloured formazans. Ephedrine and phenylephrine can be determined simultaneously by applying the proportional-equation method to the reaction rate and absorbance amplitude of the kinetic curves obtained for each analyte. The calibration graph was linear over the range 0.7–70.0 g mL^–1 ephedrine and 1.2–70.0 g mL^–1 phenylephrine. The proposed method was applied to the determination of ephedrine in pharmaceutical samples with a mean recovery of 103.2%. Synthetic mixtures of ephedrine and phenylephrine in ratios between 3:1 and 1:5 were satisfactorily resolved.     

Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(?)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid?Cliquid extraction of liver microsomal samples with methyl tert-butyl ether, EPH and PEPH were separated on a C₁₈ reversed-phase column (200 mm × 4.6 mm, 5 ??m) with methanol?C0.5% sodium dodecyl sulfate?Cphosphoric acid?Ctriethylamine 60:40:1.25:1 (v/v) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5?C60.0 ??g mL^?1, the limit of quantification was 1.5 ??g mL^?1, and intra-day and inter-day variability was <10.0%. Average extraction recovery of the two analytes was >73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and ??-naphthoflavone.