Acetonitrile Water Research Articles

Simultaneous determination of tazarotene, clindamycin phosphate and their active metabolites in Bama mini-pig skin by LC-MS/MS: Application to the development of a tazarotene/clindamycin phosphate cream

Topical tazarotene combined with clindamycin phosphate can significantly improve the adherence and outcomes for the treatment of acne vulgaris than monotherapy, a novel tazarotene (0.05%)/clindamycin phosphate (1.2%) cream is thus developed. However, the pharmacokinetics and potential interaction of tazarotene and clindamycin phosphate in skin when formulated together remain unknown, which should be investigated to assess this novel cream. In the present work, a sensitive and rapid LC-MS/MS method for simultaneous determination of tazarotene, clindamycin phosphate and their active metabolites tazarotenic acid, clindamycin in Bama mini-pig skin was developed and reported for the first time. After pretreatment of the skin samples, the analytes were well separated on a Hypersil BDS C8 column (4.6 × 100 mm, 2.4 μm) using 0.2% (v/v) formic acid-0.1% (w/v) ammonium acetate water solution and acetonitrile as mobile phase in linear gradient elution. Quantification of tazarotene, clindamycin phosphate and their active metabolites tazarotenic acid, clindamycin was conducted under positive electrospray ionization mode using multiple reactions monitoring detection. The LC-MS/MS method was fully validated and then applied to the dermal pharmacokinetic study of the tazarotene/clindamycin phosphate cream. According to the obtained results, tazarotene and clindamycin phosphate did not have any drug-drug interaction when they were formulated together in the cream for topical application. Their absorption and metabolism features in the skin were also characterized, which can support the clinical medication regimen of tazarotene/clindamycin phosphate cream.

HPLC-UV analytical validation of a method for quantification of progesterone in ex vivo trans-corneal and trans-scleral diffusion studies

Progesterone (PG) diminishes free radical damage and thus can afford protection against oxidative stress affecting the retina. The therapeutic use of PG is limited because it is a highly hydrophobic steroid hormone with very low solubility in water. This is the main drawback for the therapeutic application of PG at ocular level. The aims of this study were: (i) to analyze if PG causes ocular irritation (ii) to validate a HPLC method to determine PG in ex vivo studies and (iii) to evaluate PG permeation through cornea and sclera. A high performance liquid chromatographic method was developed and validated to detect PG incorporated to β-cyclodextrin using a Waters Sunfire C18 (150 × 4.6 mm) reverse-phase column packed with 5 μm silica particles using a mobile phase consisted of a mixture of acetonitrile (ACN) and pure water 80:20 (v/v), pH 7.4. The limit of detection and the limit of quantification for 50 μL injection of PG were found to be 0.42 and 1.26 μg/mL, respectively. The calibration curve showed excellent linearity over the concentration range (0.5 μg/mL to 100 μg/mL). As proof of concept, ex-vivo experiments to investigate PG permeation through cornea and sclera with vertical diffusion cells were carried out to quantify PG diffusion. Ex vivo experiments demonstrate its applicability to investigate permeation levels of PG from 6.57 ± 0.37 μg/cm2 at cornea and 8.13 ± 0.85 μg/cm2 sclera. In addition, at the end of diffusion studies the amount of PG retained in each tissue was also quantified, and it was 40.87 ± 9.84 μg/cm2 (mean ± SD; n = 6) in cornea and 56.11 ± 16.67 μg/cm2 (mean ± SD; n = 6) in sclera.

DEVELOPMENT AND VALIDATION OF NEW STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF ELBASVIR AND GRAZOPREVIR IN BULK AND PHARMACEUTICAL DOSAGE FORM

The present study describes the development and validation of a new stability indicating RP-HPLC method for simultaneous estimation of elbasvir and grazoprevir in bulk and pharmaceutical dosage form. The chromatographic separation was done by using reverse phase YMC column (4.6 x 250mm, 5μm).The mobile phase used was mixture of HPLC grade water acetonitrile (70:30V/V) at flow rate of 1ml/ min in isocratic mode and detection was carried out using PDA detector at 244nm.The developed method showed a good linearity in the range of 100-500μg/mL for elbasvir and 200-1000 μg/mL for grazoprevir with regression co-efficient of 0.999 for both the drugs. The % recovery of drugs was found to be 100.1% for elbasvir and 100.38% for grazoprevir. The proposed stability indicating method was accurate, precise, robust, stable and specific. The developed method was validated in accordance with ICH guidelines.

Rapid Determination of Total Tryptophan in Yoghurt by Ultra High Performance Liquid Chromatography with Fluorescence Detection.

Tryptophan (TRP) is an essential amino acid which cannot be synthesized by humans and animals, but has to be supplied by exogenous sources, notably through the diet. The bulk of dietary TRP flows into the synthesis of body’s proteins, but the TRP metabolism also involves several biochemical reactions (i.e., serotonin and kynurenine pathways). Defects in the TRP transport mechanism or catabolism are related to a large number of clinical abnormalities. Therefore, dietary TRP intake is necessary not only for the body’s growth but also for most of the body’s metabolic functions. Among protein-based foods, milk proteins provide a relatively high amount of TRP. In this paper, a rapid chromatographic method for TRP determination in yoghurt, by ultra high performance liquid chromatography on a reversed-phase column with fluorescence detection (280 nm Ex; 360 nm Em), is provided. A linear gradient elution of acetonitrile in water allowed TRP analysis in 8.0 min. The limit of detection and limit of quantification of the method were 0.011 ng/µL and 0.029 ng/µL, respectively, using 5-methyl-l-tryptophan as the internal standard. The analytical method was successfully applied to commercial yoghurts from different animal species, and the TRP values ranged between 35.19 and 121.97 mg/100 g (goat and cow Greek type yoghurt, respectively).

Determination of 2,4-Dichlorophenoxyacetic Acid in Water and Edible Seeds Samples Using Salt-Assisted Liquid-Liquid Extraction Coupled with High-Performance Liquid Chromatography

A simple, fast, and effective salt-assisted liquid-liquid extraction (SALLE) technique coupled with high-performance liquid chromatography (HPLC) was developed for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) in water and edible seeds samples. Due to the polar nature of the 2,4-D molecules, LC analysis was performed on a CN stationary phase by using acetonitrile and acidic water as mobile phase. Extraction was achieved based on salting-out phenomenon. Affecting variables on the extraction efficiency of the 2,4-D, such as the solvent type and its volume, type of salt and its concentration, vortex time, and sample pH, have been investigated and optimized. The target analyte was successfully extracted by acetonitrile and sodium chloride as extracting solvent and salting-out agent, respectively. Under the optimized experimental conditions, calibration curve shows good linearity (r2 > 0.9957) and precision (RSD < 6.5%) in the concentration ranges of 1.0–10,000 ng/mL. The limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 1.0 ng/mL, respectively. The recoveries were in the range of 82.0–95.0 % with RSD values less than 7.2%. The method was successfully applied for the extraction and determination of 2,4-D in several edible seeds and water samples.

Simultaneous determination of β-sitosterol and gallic acid in Nigella Sativa seeds using reverse phase high performance liquid chromatography

HPLC determination of β-Sitosterol and Gallic acid in seeds of Nigella sativa was carried out using reversed-phase Grace C18 column (250 mm × 4.6 mm id, 5 µm particle size), with UV detection at 210 nm. The mobile phase for column consisted of 60:40 v/v acetonitrile–water at a flow rate of 0.5 ml/min. The pH of the mobile phase was adjusted to 3.00, with 0.05% ortho-phosphoric acid. The linear response was examined in the concentration range of Gallic acid and β-Sitosterol between 0.5 and 2.5 μg/mL, and linear correlation coefficients of 0.9983 for Gallic acid, and 0.9993 for β-sitosterol, respectively, were observed. The detection limit was 4.8 µg/mL, and the recovery test in the determination of β-Sitosterol and Gallic acid revealed 98–101% for both compounds.

Smart TLC–densitometric methods for determination of ophthalmic ternary mixture containing chloramphenicol in the presence of its synthetic precursor: Comparative eco-scaling for greenness assessment

Green analytical methods have gained a growing interest in the field of pharmaceutical research to reduce impacts on the environment and enhance analysts’ health safety. Chloramphenicol (CHL), dexamethasone sodium phosphate (DSP) and tetrahydrozoline HCl (THZ) form an ophthalmic ternary mixture that is co-formulated for conjunctivitis treatment. In the present work, for time saving and higher sensitivity, two green thin-layer chromatography (TLC) methods were developed for the determination of this ophthalmic ternary mixture in the absence or presence of p-nitroacetophenone (PNA), a synthetic precursor of chloramphenicol. In both proposed methods, silica gel 60 F254 plates were used as the stationary phase. The mobile phase used for method (A) was ethanol‒water‒ammonia (7.0:2.5:0.5, V/V), while, for method (B), acetonitrile‒water‒ammonia (10.0:3.0:0.5, V/V) was used as the mobile phase. TLC separation was followed by quantitative determination of the aforementioned drugs at wavelengths 242.0 nm and 220.0 nm. Both methods were validated in compliance with the International Conference on Harmonisation (ICH) guidelines, where both methods were found to be reliable, reproducible, and selective. Statistical comparison of the developed methods was done with a reported high-performance liquid chromatography (HPLC) method where no significant difference was found. Analytical eco-scaling depends on penalty point which was calculated to be 92, 88 and 87 for methods A, B and the reported HPLC, respectively, suggesting that the proposed methods are eco-friendlier with penalty point scoring very high on the scale than the reported one.

Naked eye detection of moisture in organic solvents and development of alginate polymer beads and test cassettes as a portable kit

New dabsyl-thiophene based receptor DABT and its mercury complex DABT-Hg is reported as a colorimetric sensor for rapid and sensitive detection of trace amount of water in aprotic solvents. Based on intramolecular charge transfer in the excited state, the receptor dabsyl-thiophene (yellow color) binds with the mercury ions (magenta color) to stimulate a colorimetric response. The mercury complex is used as a moisture sensor in THF, acetone, and acetonitrile due to its instability in moisture containing organic solvents. The probe exhibits higher sensitivity towards water in THF (LOD = 0.0041% w/w), acetone (LOD = 0.0144% w/w) and acetonitrile (LOD = 0.1008% w/w). The dissociation of mercury from probe DABT-Hg in the presence of water is accountable for the colorimetric response as proven by the 1H NMR and ESI-MS studies. DABT-Hg is the first mercury based complex for the detection of moisture in organic solvents. Test paper strip and PVA thin film doped with the probe were successfully used to detect moisture content in organic solvents. DABT-Hg incorporated alginate beads are prepared to determine the water content in triethylamine and ethylene glycol. Portable test cassettes are developed for the on-site detection of distilled and undistilled wet solvents in the chemical laboratory through naked-eye detection.

Simultaneous determination of twelve quinones from Rubiae radix et Rhizoma before and after carbonization processing by UPLC-MS/MS and their antithrombotic effect on zebrafish

Rubiae Radix et Rhizoma (called “Qiancao”, QC), the root and rhizome of Rubia cordifolia L., has been widely used in clinical practice for its excellent performance in removing blood stasis and haemostasis. However, after carbonization processing, significant changes occurred in chemical components of the charcoal of Rubiae Radix et Rhizoma (called “Qiancaotan”, QCT), which enhanced the performance in haemostasis and weakened the performance in removing blood stasis in clinic. In order to study the material basis of function variation during processing, a rapid, reliable, accurate and validated UPLC–MS/MS approach was established to determine twelve quinones in QC and QCT simultaneously. Meanwhile, the antithrombotic effect of target components on zebrafish thrombus model induced by phenylhydrazine (PHZ) was investigated. Chromatographic separation was accomplished on an ACQUITY UPLC C18column with acetonitrile–water containing 0.2 % (v/v) formic acid as mobile phase, at a flow rate of 0.30 mL/min. Quantitation was performed using multiple reaction monitoring (MRM) in positive and negative ion electrospray ionization (ESI). Furthermore, the activity evaluation studies showed that the reduction of removing blood stasis effect of QCT was due to the decrease of dehydro-α-lapachone, lapachol, rubioncolin C and mollugin. This study demonstrated that the method has been successfully applied to determine the content of twelve quinones responsible for the function variation of QCT, and provided a new insight into the material basis and the effect of eliminating stasis before and after processing of QC.

Simple Iron(III) Complex Based Highly Sensitive Fluorescent Off‐On Sensor for the Detection of Trace Amount of Water in Organic Solvents and Edible Oilseeds

AbstractMoisture detection in organic solvents and materials by using chemical sensors represent the latest research area in analytical chemistry. In this paper, we report a new fluorescent chemical sensor for the sensitive detection of trace amount of water in Tetrahydrofuran(THF), acetone and acetonitrile. The new probe L.Fe(III) complex consists of the fluorescent reporter dansyl dye attached with Fe(III) ion. Ligand L, and probe L.Fe(III) exhibits solvatochromism by intramolecular charge transfer (ICT) due to the donor‐acceptor properties of the dansyl compound. Probe L.Fe(III) is a non‐fluorescent compound and it becomes highly fluorescent in the presence of trace amount of water in THF, acetone and acetonitrile. The probe shows highly sensitive towards the water in THF with a limit of detection (LOD) as low as 0.0003 weight(wt) %. 1H‐NMR, ESI‐MS and fluorescence lifetime studies established that displacement of metal from the probe is responsible for fluorescent enhancement. Further, for the first time probe L.Fe(III) has been explored as a fluorescence indicator for the qualitative and quantitative detection of trace‐level of water content in various edible oilseeds such as groundnut, mustard, sunflower, sesame and almond.

Synthesis of Molecularly Imprinted Polymer and Its Application as Solid-Phase Extraction Sorbent for Ceftazidime Determination in Human Serum and Urine Samples

A novel molecularly imprinted solid–phase extraction method was developed coupled with HPLC for ceftazidime determination in biological fluids. Various parameters such as molecular recognition properties, adsorption capacity and selectivity of the molecularly imprinted polymer (MIP) were evaluated. The optimal conditions for solid-phase extraction consisted of conditioning with 5 mL of methanol and 20 mL of deionized water at neutral pH, aqueous sample loading (50 mL at pH 3.0), washing using 5 mL of acetone and elution with 5 mL of acetonitrile–water solution (2 : 8, v/v). The MIP selectivity was evaluated by checking several substances with molecular structures similar to that of ceftazidime. The MIP relative standard deviation was 4.1% (n = 5). The limits of detection and quantification for ceftazidime were 7 and 25 μg/L, respectively, while linearity range was 25–800 μg/L.

Effects of Analyte Concentration on the Protonation Sites of 4-Aminobenzoic Acid upon Atmospheric Pressure Chemical Ionization As Revealed by Gas-Phase Ion-Molecule Reactions.

The most basic site of 4-aminobenzoic acid in aqueous solution is the amino nitrogen, while the carbonyl oxygen is calculated to be the most basic site in the gas phase. However, the preferred protonation site of 4-aminobenzoic acid upon electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) depends upon the ionization solvent and ion source parameters. The influence of the concentration of the analyte on the manifested protonation sites upon APCI has not been investigated and is reported here. Gas-phase ion-molecule reactions of trimethoxymethylsilane were used to identify the protonation sites of 4-aminobenzoic acid ionized using APCI with methanol or acetonitrile-water as the solvent. The nitrogen-protomer was found to be about twice as abundant as the oxygen-protomer at low analyte concentrations (10-9-10-6 M) in methanol solvent. This finding was rationalized on the basis of a previous finding that when the O-protomer is surrounded by more than eight methanol molecules in the gas phase it starts behaving as if it were in an aqueous solution and converts to the N-protomer. At greater analyte concentrations (≥10-4 M), the amino group was predominantly protonated, which was rationalized based on the formation of a particularly stable proton-bound dimer of 4-aminobenzoic acid that preferentially dissociates to form the N-protomer. The above findings suggest that solution processes are much more important in APCI than commonly assumed, in agreement with recent literature. Indeed, when 1:1 (v/v) acetonitrile-water was used as the solvent system for 4-aminobenzoic acid, the N-protomer was predominantly generated at all analyte concentrations.

Development and validation of an analytical method for quantification of potential genotoxic impurity 4‐dimethylaminopyridine in linagliptin active pharmaceutical ingredient using Hydrophilic Interaction Liquid Chromatography

AbstractA sensitive analytical method was developed to estimate the potential genotoxic impurity 4‐dimethylaminopyridine in linagliptin pharmaceutical ingredient. 4‐Dimethylaminopyridine is a reactive reagent used in synthesis of linagliptin. Sensitivity of the studied method was excellent at a level of 30 ppm of 4‐dimethylaminopyridine relative to linagliptin when samples of linagliptin were prepared at 10.0 mg/mL. A silica (150 mm × 4 mm, 3 µm) column was used to analyze the samples in hydrophilic interaction liquid chromatography mode. A mobile phase of water–acetonitrile (15:85, v/v) containing 10.0 mM ammonium acetate was prepared and adjusted to pH 6.2. Flow rate of the method was 1.3 mL/min and the ultraviolet detector was set at 258 nm. This method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines in terms of detection limit, quantitation limit, linearity, precision, accuracy, specificity, and robustness. The method had excellent linearity in the concentration range of 15–450 ppm with a correlation coefficient of 0.999. Detection limit and quantitation limit were 4.46 and 14.87 ppm, respectively.

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

In recent years, short peptide self-assembled materials, prepared under the control of the thermolysin catalyst, have been investigated extensively and shown to acquire various morphologies and fun...

Determination of Rufinamide in the Presence of 1-[(2,6-Difluorophenyl)Methyl]-1H-1,2,3-Triazole-4 Carboxylic Acid Using RP-HPLC and Derivative Ratio Methods as Stability Indicating Assays to Be Applied on Dosage Form.

Rufinamide is a triazole derivative that is structurally dissimilar to other marketed antiepileptic drugs, has been assumed a marketing authorization, by the European Union and FDA, for use as a complementary therapy for seizures associated with Lennox-Gastaut syndrome. This work is concerned with development of two methods for determination of rufinamide (RUF) in presence of 1-[(2,6-difluorophenyl)methyl]-1H-1,2,3-triazole-4 carboxylic acid as its alkaline degradation product in dosage form. The first method was capable of determing RUF in the presence of its alkaline degradation product and in dosage form. Kromasil C8 column and mobile phase consisting of acetonitrile-water (50:50, v/v) were used and UV detection at 210 nm. In the second method, first derivative ratio spectrophotometry, RUF was determined by measuring peak amplitude at 269.5 nm over 5-30 μg/mL. The linearity range of RUF was 10-90 μg/mL for HPLC method covering its therapeutic range with r2 = 0.9999. Forced degradation under alkaline conditions was carried out, the degradation product was isolated and its structure was confirmed. Both methods were validated in accordance to ICH guidelines. Statistical analysis revealed no significant difference between obtained results and reported ones. The present study is useful for therapeutic drug monitoring and routine analysis of RUF in quality control laboratories. Kinetics of the alkaline degradation of RUF was studied by following the concentration of the remaining drug until complete degradation was achieved.

Determination of sirolimus in blood by high-performance liquid chromatography with ultraviolet detection - Experience and review

Sirolimus (SRL) is a potent immunosuppressant. Therapeutic drug monitoring (TDM) of SRL is required to optimize the therapy. Immunoassay, high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and HPLC with mass-spectrometric detection (HPLC-MS or HPLC/MS/MS) have been used in the analysis of SRL. The purpose of this study was to share our experience in validating a HPLC-UV method for analyzing SRL and to have an overview of HPLC-UV methods used in SRL assay. A validated HPLC/UV method developed by Wyeth-Ayerst Research with minor modification was use to determine SRL concentration in human whole blood. An Alltima C18 column (5 μm, 150 × 2.1 mm) was used as the stationary phase. The mobile phase was 60% acetonitrile in water, and the flow rate was 0.5 mL/min. Samples were prepared by spiking human whole blood (0.5 mL) with the internal standard (IS) and designated amount of SRL, except blank. Zinc sulfate (50 g/L, 1 mL) and acetone (1 mL) were used for hemolysis and deproteinization. After alkalizing supernatant with 100 mM NaOH (0.2 or 0.3 mL), 1-chlorobutane (2 mL) was used for extraction. The 1-chlorobutane layer was dried, reconstituted with mobile phase and back extracted with 0.5 mL of n-hexane. The limitation of quantification was 2.5 ng/mL and the standard curve was linear at the concentration range of 2.5-75 ng/mL. The intraday and interday coefficients of variation were 2.1-5.2% and 2.8-5.7%, respectively. The intraday and interday relative errors were -0.03-5.3% and 0.7-3.3%, respectively. The recoveries for SRL and the IS were 78.5-92.8% and 76.9 ± 3.9%, respectively. The samples were proven to be stable after 3 freeze/thaw cycles, and the extract was stable over 24 hr at an autosampler set to 4°C. In addition to an overview of the chemical properties of SRL, different HPLC-UV methods for the quantification of SRL were provided to identify analytic parameters that are critical for the establishment of HPLC-UV assay for SRL.

Bioassay-Guided Separation of Centipeda minima Using Comprehensive Linear Gradient Centrifugal Partition Chromatography

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n-hexane–acetonitrile–water (10:2:8, v/v), ethyl acetate–acetonitrile–water (10:2:8, v/v), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v). The lower phase of the n-hexane–acetonitrile–water (10:2:8, v/v) was used as the stationary phase, while its upper phase, as well as ethyl acetate–acetonitrile–water (10:2:8), and water-saturated n-butanol–acetonitrile–water (10:2:8, v/v) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima, using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

A Rapid and Sensitive HPLC Method for Simultaneous Determination of Irinotecan Hydrochloride and Curcumin in Co-delivered Polymeric Nanoparticles.

In recent years, a great deal of attention has been paid to the combined use of multiple antitumor drugs for better cancer treatment. The aims of the study are to construct a nanoparticle drug delivery system for the co-delivery of irinotecan hydrochloride and curcumin and to develop an analytical method for simultaneously quantifying these molecules, which is essential for further studies of the co-delivered nano system. The irinotecan hydrochloride and curcumin co-delivered nanoparticle (ICN) were prepared by combinatorially entrapping them into polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA) polymeric nanoparticles. A simple, sensitive and rapid high-performance liquid chromatography method was developed and validated to simultaneously quantify the compounds in the co-delivered nanoparticle system. Acetonitrile and ultrapure water containing sodium dodecyl sulfate (0.08mol/L), disodium phosphate (Na2HPO4, 0.002mol/L) and acetic acid (4%, v/v) were used as the mobile phase and their ratio was set at 50:50. The flow rate was set to 1.0mL/min, and the temperature in the column oven was maintained at 40°C. The analysis was carried out at 256 and 424nm to assess irinotecan hydrochloride and curcumin, respectively. Detectors with only one channel can also visualize both analytes in one chromatogram at 379nm and still demonstrate acceptable sensitivity. The retention times for irinotecan hydrochloride and curium were 3.317 and 5.560min, respectively. The method developed was confirmed to be sensitive, accurate (recovery, 100±2%), precise (relative standard deviation, RSD≤1%), robust and linear (R2≥0.9996) in the range from 2.05 to 1050μg/mL. The presented method has been used to quantify irinotecan hydrochloride and curcumin in the co-delivered ICN nano system to assess the drug delivery quality of the nanoparticles and can also be used for routine analysis because of its simplicity and accuracy.

Pharmacokinetic Effects of l-Tetrahydropalmatine on Ketamine in Rat Plasma by Ultraperformance Liquid Chromatography Tandem Mass Spectrometry.

Male Sprague-Dawley rats (n = 18) were randomly divided into three groups: a saline group (20 mL/kg by gavage), a ketamine (KET) group (100 mg/kg by gavage), and a KET (the same routes and doses) combined with levo-tetrahydropalmatine (l-THP; 40 mg/kg by gavage) group (n = 6). Blood samples were acquired at different time points after drug administration. A simple and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to determine the concentrations of KET and its metabolite, norketamine (NK), in rat plasma. Chromatographic separation was achieved using a BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with chlorpheniramine maleate (Chlor-Trimeton) as an internal standard (IS). The initial mobile phase consisted of acetonitrile–water with 0.1% methanoic acid (80 : 20, v/v). The multiple reaction monitoring (MRM) modes of m/z 238.1→m/z 179.1 for KET, m/z 224.1→m/z 207.1 for NK, and m/z 275→m/z 230 for Chlor-Trimeton (IS) were utilized to conduct a quantitative analysis. Calibration curves of KET and NK in rat plasma demonstrated good linearity in the range of 2.5–500 ng/mL (r > 0.9994), and the lower limit of quantification (LLOQ) was 2.5 ng/mL for both. Moreover, the intra- and interday precision relative standard deviation (RSD) of KET and NK were less than 4.31% and 6.53%, respectively. The accuracies (relative error) of KET and NK were below -1.41% and -6.07%, respectively. The extraction recoveries of KET and NK were more than 81.23 ± 3.45% and 80.42 ± 4.57%, respectively. This sensitive, rapid, and selective UPLC-MS/MS method was successfully applied to study the pharmacokinetic effects of l-THP on KET after gastric gavage. The results demonstrated that l-THP could increase the bioavailability of KET and promote the metabolism of KET. The results showed that l-THP has pharmacokinetics effects on KET in rat plasma.

Efficient Preparation of Bafilomycin A1 from Marine Streptomyces lohii Fermentation Using Three-Phase Extraction and High-Speed Counter-Current Chromatography.

An efficient strategy was developed for the rapid separation and enrichment of bafilomycin A1 (baf A1) from a crude extract of the marine microorganism Streptomyces lohii fermentation. This strategy comprises liquid−liquid extraction (LLE) with a three-phase solvent system (n-hexane–ethyl acetate–acetonitrile–water = 7:3:5:5, v/v/v/v) followed by separation using high-speed counter-current chromatography (HSCCC). The results showed that a 480.2-mg fraction of baf A1-enriched extract in the middle phase of the three-phase solvent system was prepared from 4.9 g of crude extract after two consecutive one-step operations. Over 99% of soybean oil, the main hydrophobic waste in the crude extract, and the majority of hydrophilic impurities were distributed in the upper and lower phase, respectively. HSCCC was used with a two-phase solvent system composed of n-hexane–acetonitrile–water (15:8:12, v/v/v) to isolate and purify baf A1 from the middle phase fraction, which yielded 77.4 mg of baf A1 with > 95% purity within 90 min. The overall recovery of baf A1 in the process was determined to be 95.7%. The use of a three-phase solvent system represents a novel strategy for the simultaneous removal of hydrophobic oil and hydrophilic impurities from a microbial fermentation extract.