Simple LC Research Articles

LC–DAD–ESI-MS–MS Separation and Chemical Characterization of the Inflammatory Fraction of the Roots of Euphorbia kansui

In China the roots of Euphorbia kansui T.N. Liou ex T.P. Wang, known as ‘kansui’, have been used for centuries as a herbal remedy for edema, ascites, and asthma. Kansui, has inflammatory and tumor-promoting toxicity, and other side-effects, however, which have seriously restricted its clinical application. In the work discussed in this paper a simple and rapid LC–DAD–ESI-MS–MS method has been established for separation and characterization of the main compounds in the toxic fraction of E. kansui roots. Twelve diterpene derivatives were identified in the inflammatory fraction of kansui: kansuinine C, kansuinine B, kansuinine A, kansuinine D, 5-O-benzoyl-20-deoxyingenol, kansuinine E, kansuiphorin C, 3-O-benzoyl-20-deoxyingenol, 3-O-(2′E,4′Z-decadienoyl)-5-O-acetylingenol, 3-O-(2′E,4′Z-decadienoyl)-20-deoxyingenol, 20-O-(2′E, 4′E-decadienoyl)ingenol, and 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol. The inflammatory fraction was separated on a C18 reversed-phase column with a mobile phase gradient. The proposed method is a scientific and technical platform enabling the herbal medicine industry to perform quality control and ensure the safety of preparations that contain this class of poisonous diterpenoids.

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC–MS

A simple and sensitive LC–MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2–200 ng/mL for DUL, OLA, and STN, 4–200 ng/mL for DSTN, 5–1000 ng/mL for ARI, DARI and finally 2–1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

Numerical Study of Low-Loss Cross Left-Handed Metamaterials at Visible Frequency

We present a numerical study of left-handed metamaterials (LHMs) composed of double cross pairs at visible frequency. The S-parameter retrieval method is adopted to confirm the negative refractive characteristic of this design. Compared with fishnet LHMs, the proposed cross LHMs have a much lower loss, which will greatly facilitate practical applications. It is also found that, with the same size of resonant cells, the cross LHMs have a higher frequency than fishnet LHMs, which is explained using a simple effective LC circuit model.

An LC–MS–MS Method for the Simultaneous Quantitation of Acyclovir and Valacyclovir in Human Plasma

A simple, sensitive and selective LC–MS–MS method has been developed for the simultaneous determination of acyclovir and valacyclovir in human plasma. Acyclovir and valacyclovir in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of 0.1% formic acid: methanol (30:70% v/v). The method was validated over a linear range of 47–10,255 and 5–1,075 ng mL−1 for acyclovir and valacyclovir respectively. The LOQs were 47.6 and 5.0 ng mL−1. The validated method was applied for the quantitation of acyclovir and valacyclovir from plasma samples in a pharmacokinetic study.

Development and validation of an improved liquid chromatography–mass spectrometry method for the determination of pemetrexed in human plasma

An improved liquid chromatography–mass spectrometry method for the determination of pemetrexed in human plasma was developed and validated using a simple quadrupole LC–MS and a new SPE cartridge (Plexa ® Bond Elut). The analysis was achieved with a C18 analytical column using a mobile phase consisting of formic acid/acetonitrile and isocratic flow for 7 min. The linear ranges ( r 2 > 0.99) were found from 5 to 5000 ng/mL. The lower limit of detection was 2.5 ng/mL. Within-day and between-day precisions were less than 7.2% and inaccuracy did not exceed 2.8%. This new method is suitable to support pharmacokinetic studies and drug monitoring.

Analysis of trace levels of domoic acid in seawater and plankton by liquid chromatography without derivatization, using UV or mass spectrometry detection

Quantitation of trace levels of domoic acid (DA) in seawater samples usually requires labour-intensive protocols involving chemical derivatization with 9-fluorenylmethylchloroformate and liquid chromatography with fluorescence detection (FMOC–LC–FLD). Procedures based on LC–MS have been published, but time-consuming and costly solid-phase extraction pre-concentration steps are required to achieve suitable detection limits. This paper describes an alternative, simple and inexpensive LC method with ultraviolet detection (LC–UVD) for the routine analysis of trace levels of DA in seawater without the use of sample pre-concentration or derivatization steps. Qualitative confirmation of DA identity in dubious samples can be achieved by mass spectrometry (LC–MS) using the same chromatographic conditions. Addition of an ion-pairing/acidifying agent (0.15% trifluoroacetic acid) to sample extracts and the use of a gradient elution permitted the direct analysis of large sample volumes (100 μl), resulting in both high selectivity and sensitivity (limit of detection = 42 pg ml −1 by LC–UVD and 15 pg ml −1 by LC–MS). Same-day precision varied between 0.4 and 5%, depending on the detection method and DA concentration. Mean recoveries of spiked DA in seawater by LC–UVD were 98.8% at 0.1–10 ng ml −1 and 99.8% at 50–1000 ng ml −1. LC–UVD exhibited strong correlation with FMOC–LC–FLD during inter-laboratory analysis of Pseudo-nitzschia multiseries cultures containing 60–2000 ng DA ml −1 ( r 2 > 0.99), but more variable results were obtained by LC–MS ( r 2 = 0.85). This new technique was used to confirm the presence of trace DA levels in low-toxicity Pseudo-nitzschia spp. isolates (0.2–1.6 ng ml −1) and in whole-water field samples (0.3–5.8 ng ml −1), even in the absence of detectable Pseudo-nitzschia spp. cells in the water column.

RP-LC Simultaneous Determination of Nebivolol Hydrochloride and Amlodipine Besilate in Bi-Layer Tablets

A simple and rapid LC method was developed and validated for simultaneous estimation of nebivolol and amlodipine in a bi-layer tablet formulation. Efficient chromatographic separation was achieved on (USP L10) Hypersil BDS cyano, 5 μm, 250 mm × 4.6 mm column with simple mobile phase composition delivered in isocratic mode. The method had requisite accuracy, selectivity, sensitivity, robustness and precision to assay nebivolol and amlodipine in pharmaceutical dosage form. Degradation products resulting from the stress studies did not interfere with the detection of nebivolol and amlodipine, these peaks remained pure and thus proved to be stability indicating. The mass balance of the stressed sample was in the range 99.0–100.2% for amlodipine and 99.3–100.3% for nebivolol.

Determination of Boanmycin in Pharmaceutical Preparations by a Simple and Rapid Ion-pair LC Method

An ion-pair HPLC method with UV detection is described for the determination of boanmycin in freeze-dried pharmaceutical preparations. Chromatographic separation was achieved on a 5 μm Agilent Eclipse XDB-C18 column (150 × 4.6 mm). The mobile phase contained methanol, acetonitrile, 055 M sodium hexanesulfonate and 0.08 M acetic acid in water. UV detection was at 291 nm. The assay was validated over a linear concentration range from 0.05 to 120 μg mL−1. The method was applied to determine the content of BAM in freeze-dried powders.

Validated Optimized Method for Simultaneous Analysis of Active Silymarin Components and Dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene Dioxybiphenyl-2,2′-dicarboxylate in a Pharmaceutical Preparation by Use of a Monolithic Silica C18 Column

A simple, rapid, and sensitive isocratic reversed-phase LC method using a monolithic column has been developed and validated for simultaneous analysis of the active components of silymarin [taxifolin, silydianin, silychristin, diastereomers of silybin (silybin A and B), and diastereomers of isosilybin (isosilybin A and B)] and dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate in a commercial formulation. The mobile phase was a 45:55 (v/v) mixture of methanol and 5 mM NaH2PO4 (adjusted to pH 2.75 with phosphoric acid) at a flow rate of 1.5 mL min−1. UV detection was performed at 288 nm and quantification was based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness.

Development and validation of an LC method for the determination of emtricitabine and related compounds in the drug substance

A robust, precise, sensitive, linear, and simple RP LC method coupled with UV for the determination of emtricitabine or 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC) and its related substances is described. The method uses an RP C18 column (25 cm x 4.6 mm i.d.), 5 microm kept at a temperature of 35 degrees C. The mobile phases for gradient elution consist of ACN, phosphate buffer (pH 4.4), and water. The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. A system suitability test (SST) was developed to verify the adequate performance of the chromatographic system. The developed method was further validated with respect to robustness, precision, sensitivity, and linearity. A central composite design was applied to examine the robustness of the method. The method shows good precision, sensitivity, linearity, and robustness. Three commercial FTC samples were examined using this method. This method is suitable to be used for the determination of related substances and assay of FTC.

LC Evaluation of Intestinal Transport of Praziquantel

Praziquantel (PZQ) is a highly lipophilic drug with low aqueous solubility. Despite this, it is well absorbed from the gastrointestinal tract. In this study, a simple LC method was developed and validated, in order to monitor the concentration of PZQ in TC-199 buffer in vitro, in the rat everted gut sac absorption model. PZQ was analyzed by a reversed-phase LC method with an isocratic mobile phase containing acetonitrile and water in the proportions 45:55. The flow-rate was 1 mL min−1 and PZQ was determined by measuring absorbance at 215 nm, at 25 °C. The method was found to be specific, as none of the components of TC-199 or intestinal sac artefacts interfered with the drug peak. Recovery was within acceptable statistical limits. The limit of detection was 0.54 μg mL−1 and the limit of quantitation was 1.63 μg mL−1. The calibration curve was found to be linear in the concentration range of 10–90 μg mL−1 PZQ. The proposed method was found to be rapid and selective and hence can be applied in the monitoring of the absorption of PZQ in in vitro everted gut sac absorption studies.

Determination of Pitavastatin in Human Plasma by LC–MS–MS

A simple and rapid LC–MS–MS assay was developed and validated for the quantitative determination of pitavastatin in human plasma. Sample pretreatment involved simple protein precipitation by addition of acetonitrile. Separation was on an Agilent 1.8 μm Zorbax SB-C18 column (150 mm × 4.6 mm) at 25 °C using isocratic elution with methanol–0.1% formic acid in water (85:15, v/v) at a flow rate of 0.4 mL min−1. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 422.0 → 290.1 for pitavastatin, and m/z 330.1 → 192.1 for paroxetine (IS). LC–MS–MS was found to improve the quantitation of pitavastatin in plasma and was successfully applied in pharmacokinetic studies.

LC Assay for Ciprofloxacin Hydrochloride Ophthalmic Solution

The objective of the current study was to develop and subsequently validate a simple, sensitive and precise reversed-phase LC method for the determination of ciprofloxacin hydrochloride in ophthalmic solution form. The chromatographic separation of ciprofloxacin hydrochloride was achieved on a Symmetry Waters C18 column using UV detection at 275 nm. The optimized mobile phase consisted of 2.5% acetic acid solution: methanol:acetonitrile (70:15:15, v/v/v). The proposed method provided linear responses within the concentration range 1.0–6.0 μg mL−1 for ciprofloxacin hydrochloride. Correlation coefficient (r) for the ciprofloxacin hydrochloride was 0.9994. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 5% in all instances. No interference from any components of pharmaceutical dosage forms was observed.

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.

Simultaneous LC−UV Analysis of Mirodenafil and Its Two Main Metabolites in Rat Plasma and Urine, and in Tissue Homogenates

A simple, rapid, and reproducible isocratic reversed-phase LC method has been established for simultaneous analysis of mirodenafil and its two main metabolites, SK3541 and SK3544, in rat plasma, urine, and tissue homogenates. Samples were deproteinized with acetonitrile containing sildenafil (internal standard). The compounds were separated on a C18 column with 52:48 (v/v) 0.02 m ammonium acetate buffer (pH 6)—acetonitrile as mobile phase at a flow rate of 1.4 mL min−1. UV detection was at 254 nm and detection limits of mirodenafil, SK3541, and SK3544 in plasma were 0.03, 0.05, and 0.1 μg mL−1, respectively. The method is applicable to pharmacokinetic studies of mirodenafil and its metabolites in rats.

LC Determination of Isosorbide-5-Mononitrate in Human Plasma

A simple, sensitive, selective and cost effective LC–UV method was developed for determination of isosorbide mononitrate in human plasma using guaifenesin as an internal standard. Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert-butyl methyl ether and chromatographed on a C18 column using water and acetonitrile (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from 51.6 to 2064.4 ng mL−1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was 51.6 ng mL−1. The validated method was applied to the quantitation of isosorbide mononitrate from plasma samples in a pharmacokinetic study.

Cassette analysis of eight beta-blockers in bovine eye sclera, choroid–RPE, retina, and vitreous by liquid chromatography–tandem mass spectrometry

A simple, selective, and sensitive LC–MS/MS method was developed for the simultaneous extraction and determination of eight beta-blockers (atenolol, sotalol, nadolol, pindolol, timolol, metoprolol, betaxolol and propranolol) in various bovine eye tissues including sclera, choroid–RPE, retina, and vitreous. The analytes were extracted by liquid–liquid extraction after samples were alkalinized with 2% NaOH solution in water. The chromatographic separation was performed on a Hypersil-ODS C18 column (100 mm × 2.1 mm, 3.9 μm) using a gradient mixture of (A) 5 mM ammonium formate in water (pH 3.5 adjusted with formic acid) and (B) acetonitrile:methanol (75:25) containing 0.02% triethyl amine (pH 4.0; adjusted with formic acid) as mobile phase at a flow rate of 0.4 ml/min. The compounds were ionized in the positive electrospray ionization (ESI) mode and detected in the multiple reaction monitoring (MRM) mode. The average recoveries in all four eye tissues for all beta-blockers were >82%, except for sotalol (>51%). The matrix effect for beta-blockers ranged from 81 to 110% in the four eye tissues. This analytical method was validated and applied successfully for simultaneous quantification of the beta-blockers in sclera after tissue exposure using cassette dosing method. The calibration curve was linear in the range of 10–2000 ng/ml for all analytes, with the correlation coefficient >0.996. Intra-day and inter-day precision (% CV) was less than 15%, and accuracy ranged from 85 to 110% for all analytes. Scleral uptake was the lowest for sotalol and atenolol, two hydrophilic beta-blockers, and the highest for propranolol, a lipophilic beta-blocker.

Development and validation of an LC–MS/MS method for the quantification of ephedrines in urine

The objective of this study was to develop a simple and robust LC–MS/MS method for the quantification of ephedrine type substances in urine. Sample preparation consisted of a 10-fold dilution step of the samples into the internal standard solution (ephedrine-d 3, 4 μg/mL in water). Baseline separation of the diastereoisomers norpseudoephedrine–norephedrine and ephedrine–pseudoephedrine was performed on a C8-column using isocratic conditions followed by positive electrospray ionisation and tandem mass spectrometric detection. The mobile phase consisted of 98/2 (H 2O/ACN) containing 0.1% HAc and 0.01% TFA. Calibration curves were constructed between 2.5 and 10 μg/mL for norephedrine and norpseudoephedrine and 5 and 20 μg/mL for ephedrine, pseudoephedrine and methylephedrine. The bias ranged from −5.5 to 12% for norephedrine, −4.1 to 8.0 % for norpseudoephedrine, 0.3 to 2.1 % for ephedrine, 1.6 to 2.6 % for pseudoephedrine and 2.9 to 5.0 % for methylephedrine. Precision of the method varied between 2.8 and 10.4% for all compounds and the matrix effect was less than 15%. The applicability of the method has been checked by the analysis of 40 urine samples. The results were compared with those obtained with the common GC–NPD method. Results show a good correlation between both methods with correlation coefficients higher than 0.95 for all analytes.

An liquid chromatography–tandem mass spectrometry assay for determination of trace amount of new antifungal drug iodiconazole in human plasma

A simple, rapid and sensitive LC–MS/MS method for determination of trace amount of new antifungal drug iodiconazole in human plasma was developed (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[ N-methyl- N-(3-chlor-benzyl)amino]-2-propanol), was used as internal standard (IS). The analytes were extracted by liquid–liquid extraction with n-hexane after internal standard spiked. The separation was performed by a ZORBAX SB-C 18 column (3.5 μm, 2.1 mm × 100 mm) with an isocratic mobile phase consisting of methanol–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The lower limit of quantification (LLOQ) was 0.10 ng/mL. This method was successfully used to determine the concentration of iodiconazole in human plasma following dermal administration.

LC Method for Determination of Ginkgolic Acids in Mice Plasma and Its Application to a Pharmacokinetic Study

A sensitive and simple LC method for the quantification of ginkgolic acids in mice plasma has been developed. Following acetonitrile deproteinization, samples were separated on a SinoChrom ODS-AP C18 column. The mobile phase was 3% (v/v) acetic acid water solution–methanol (8:92, v/v) at a flow rate of 1.0 mL min−1. Detection was at 310 nm. Calibration curve was linear over the range of 0.25–50 μg mL−1 with intra- and inter-day precisions (RSD%) of less than 9.5%. The extraction recovery ranged from 87.0 to 90.2% (RSD 2.4–6.4%) for ginkgolic acids. The method was successfully applied to the pharmacokinetic study of ginkgolic acids in mice after oral dosing of 1.0 g kg−1.