A rapid and highly sensitive stability-indicating high-performance thin-layer chromatography technique for the determination of tedizolid phosphate with a classical univariate calibration

No high-performance thin-layer chromatography (HPTLC) techniques have been established for the determination of tedizolid phosphate (TDZP) in pharmaceutical products or physiological fluids. Therefore, a rapid and highly sensitive stability-indicating HPTLC technique has been developed for the determination of TDZP in commercial formulations with a classical univariate calibration. The HPTLC‒densitometry analysis of TDZP was carried out via chloroform‒methanol (90:10, V/V) mobile phase. The determination of TDZP was performed at the wavelength of 300 nm. The proposed HPTLC technique was linear in the range of 10‒2000 ng band^‒1. In addition, the method was found to be highly accurate (% recovery = 98.53‒101.74%), precise (%CV = 0.67‒0.91%), robust (%CV = 0.83‒0.86%), highly sensitive (LOD = 3.41 ng band^‒1, LOQ = 10.23 ng band^‒1) for the determination of TDZP. The proposed technique was also able to detect TDZP in the presence of its degradation products under various stress conditions and it can be considered as a stability-indicating method. The proposed HPTLC technique was applied for the analysis of TDZP in its commercial formulations. The TDZP contents of commercial tablets and injection were determined as 98.41% and 101.23%, respectively. These results suggested that the proposed HPTLC technique can be applied for the routine analysis of TDZP in its commercial products and newly established formulations.

     

Carrier-mediated hollow fiber liquid-phase microextraction for preconcentration followed by spectrophotometric determination of amoxicillin

Preconcentration followed by ultraviolet spectrophotometric determination of amoxicillin (Amox) in pharmaceuticals and water samples by using a three-phase hollow fiber microextraction technique based on carrier-mediated transport has been presented. Amox was extracted from an aqueous solution (source phase) at pH 9.0 into 1-octanol containing 5% (w/v) Aliquat-336 impregnated in the pores of a hollow fiber. It was then back-extracted into NaCl solution (pH = 4.0) which was already positioned as the receiving phase inside the lumen of the hollow fiber. The extraction took place due to the concentration gradient of the counterion between the source and the receiving phases. Under the optimized conditions, an enrichment factor of 240 and a limit of detection of 0.2 μmol L⁻¹ were obtained. The calibration curve was linear (R² = 0.9967) in the concentration range of 0.5–10.0 µmol L⁻¹ Amox. The interday relative standard deviation (n = 9) and the intraday relative standard deviation (n = 3) for 1.0 × 10⁻⁶ mol L⁻¹ Amox solution were 7.3 and 6.4%, respectively.

     

The effect of pH and ionic strength on the transport of alumina nanofluids in water-saturated porous media

Alumina nanofluids are one of the most useful nanofluids, especially for increasing the thermal conductivity. Due to importance of porous media in the improvement of heat transfer, this study investigates the transport and retention of gamma alumina/water nanofluid in the water-saturated porous media. For this purpose, alumina nanofluids were introduced to the porous media consisting of water-saturated glass beads possessing various pH values (4, 7 and 10) and different ionic strengths (0.001 M of KCl, CaCl₂, AlCl₃, K₂SO₄, CaSO₄, Al₂(SO₄)₃, K₂CO₃ and CaCO₃). Then the break through curve of each experiment was drawn and modeled by combining classical filtration theory with advection–dispersion equation. Single collector efficiency (η₀) and attachment efficiency (α) were calculated by classical filtration theory. Also curve fitting of experiments and modeling was achieved by minimizing the sum of squared residuals, to calculate retardation factor (R) and hydrodynamic dispersion coefficient (D) of each experiment. According to the results, in general, increase in pH and ionic strength will enhance the removal rate coefficient, retardation factor and retention while decreasing the steady-state break through concentration and the hydrodynamic dispersion coefficient. The opposite of this rule was observed and analyzed for aluminum salts. The lowest retention of nanoparticles at 31.04% can be related to their transport in background solution with pH = 4 [α = \(3.87 \times 10^{ - 2}\), K_att = \(3.33 \times 10^{ - 3}\) (min⁻¹), R = 3.93, D = 0.91 (cm² min⁻¹)], and the highest retention in nanoparticle content of 94.29% was observed in background solution containing CaCO₃ [α = \(14.33 \times 10^{ - 2}\), K_att = \(137.82 \times 10^{ - 3}\) (min⁻¹), R = 12.02, D = 0.62 (cm² min⁻¹)]. Therefore, chemistry of water plays an important role in transport and retention parameters. The classical filtration theory and the advection–dispersion model are able to perfectly model and quantify the parameters of the alumina nanofluid transport in saturated porous media.

     

Development and validation of a sensitive assay for the quantification of imatinib using LC/LC‐MS/MS in human whole blood and cell culture

We developed and validated a semi‐automated LC/LC‐MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back‐flushed onto the analytical column. Ion transitions [M + H]⁺ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter‐day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi‐automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

A simple, selective, precise, rapid and accurate stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of dapagliflozin and metformin in tablet dosage form. In this work, methanol–ethyl acetate–ammonium acetate (6:4:0.1, V/V) as the mobile phase and aluminum-backed TLC plates pre-coated with 250 µm layer of silica gel 60F₂₅₄ as the stationary phase were used for the estimation of dapagliflozin and metformin. The wavelength selected for detection was 220 nm. The linearity range was found to be 20–100 ng/spot (r² = 0.9985) for dapagliflozin and 500–2500 ng/spot (r² = 0.9984) for metformin. Validation of the developed method was performed as per the International Council for Harmonisation (ICH) guidelines. Stress testing of dapagliflozin and metformin was performed under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions. The chromatographic conditions successfully resolved dapagliflozin and metformin from their degradation products, formed under various stress conditions. From stress testing, dapagliflozin was found to be significantly degrading under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions, while metformin was found to be significantly degrading in acidic and alkaline degradation conditions and stable under oxidative, photolytic and dry-heat degradation conditions. Tablet dosage form of dapagliflozin and metformin was analyzed by the developed method.

     

Validation of a sensitive LC/MS/MS method for the determination of telaprevir and its R‐isomer in human plasma

The purpose of this study was to validate a reversed‐phase high‐performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R‐diastereomer (VRT‐127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir‐R was performed on a Waters XBridge^TM BEH Shield C₁₈, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir‐d11). An internal standard for the telaprevir‐R (telaprevir‐R‐d11) was also prepared by incubating telaprevir‐d11 in basic solution, which facilitated isomer inter‐conversion. The detection and quantitation of telaprevir, telaprevir‐R, telaprevir‐IS and telaprevir‐R‐IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

LC–MS/MS Method for the Simultaneous Determination of Free Urinary Steroids

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC–MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap^® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1–120 ng/mL for cortisol and cortisone, and 1–120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85–105 %, respectively. Our LC–MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11β-HSD2 and 11β-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.     

Analysis of ketamine and norketamine in hair samples using molecularly imprinted solid-phase extraction (MISPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS)

An anti-ketamine molecularly imprinted polymer (MIP) was synthesized and used as the sorbent in a solid-phase extraction protocol to isolate ketamine and norketamine from human hair extracts prior to LC-MS/MS analysis. Under optimised conditions, the MIP was capable of selectively rebinding ketamine, a licensed anaesthetic that is widely misused as a recreational drug, with an apparent binding capacity of 0.13 μg ketamine per mg polymer. The limit of detection (LOD) and lower limit of quantification (LLOQ) for both ketamine and norketamine were 0.1 ng/mg hair and 0.2 ng/mg hair, respectively, when 10 mg hair were analysed. The method was linear from 0.1 to 10 ng/mg hair, with correlation coefficients (R ²) of better than 0.99 for both ketamine and norketamine. Recoveries from hair samples spiked with ketamine and norketamine at a concentration of 50 ng/mg were 86% and 88%, respectively. The method showed good intra- and interday precisions (<5%) for both analytes. Minimal matrix effects were observed during the LC-MS/MS analysis of ketamine (ion suppression −6.8%) and norketamine (ion enhancement +0.2%). Results for forensic case samples demonstrated that the method successfully detected ketamine and norketamine concentrations in hair samples with analyte concentrations ranging from 0.2 to 5.7 ng/mg and from 0.1 to 1.2 ng/mg, respectively.     

Development of a validated high-performance thin-layer chromatography method for the standardization of an Ayurvedic formulation using berberine and ursolic acid

Giloy Tulsi tablet is an Ayurvedic preparation containing Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) and it is recommended for boosting the body’s immune response. This research work is about the marker-based standardization of this Ayurvedic preparation using high-performance thin-layer chromatography method. Standardization is based on the determination and quantification of the phytoconstituents berberine and ursolic acid present in Giloy Tulsi tablets. Separation was performed on pre-coated silica gel 60 F₂₅₄ plate as the stationary phase, with chloroform‒acetone‒formic acid (6:3.5:0.5, V/V) as the mobile phase. Identification and quantification were conducted densitometrically at 330 nm. The developed method resulted in good quality peak shape and enabled high-quality resolution of biomarkers. The R_F value for berberine (0.46 ± 0.02) and for ursolic acid (0.68 ± 0.02) in both reference standard and formulation were found to be comparable. The method was validated for specificity, linearity, limit of detection, limit of quantification, intra-day and inter-day precisions, accuracy, and robustness. The limit of detection values were 91 and 153 ng/band for berberine and ursolic acid, respectively. The limit of quantification values were 175 and 465 ng/band for berberine and ursolic acid, respectively. Regression analysis of the calibration data revealed a good linear relationship between peak area response and concentration in the range 200‒1000 ng/band for berberine (r² = 0.995) and 500‒2500 ng/band for ursolic acid (r² = 0.9968). The accuracy of the method, determined by measurement of recovery at three different levels, was in the range 98‒102% for both markers. These results are indication of reliability, reproducibility, accuracy, and precision of the method.

     

Determination of glyphosate and aminomethylphosphonic acid in aqueous soil matrices: A critical analysis of the 9‐fluorenylmethyl chloroformate derivatization reaction and application to adsorption studies

The assessment of the environmental fate of glyphosate and its degradation product (aminomethylphosphonic acid) is of great interest given the widespread use of the herbicide. Studies of adsorption–desorption and transport processes in soils require analytical methods with sensitivity, accuracy, and precision suitable for determining the analytes in aqueous equilibrium solutions of varied complexity. In this work, the effect of factors on the yield of the derivatization of both compounds with 9‐fluorenylmethyl chloroformate for applying in aqueous solutions derived from soils was evaluated through factorial experimental designs. Interference effects coming from background electrolytes and soil matrices were established. The whole method had a linear response up to 640 ng/mL (R² > 0.999) under optimized conditions for high‐performance liquid chromatography with fluorescence detection. Limits of detection were 0.6 and 0.4 ng/mL for glyphosate and aminomethylphosphonic acid, respectively. The relative standard deviation was 4.4% for glyphosate (20 ng/mL) and 5.9% for aminomethylphosphonic acid (10 ng/mL). Adsorption of compounds on four different soils was assessed. Isotherm data fitted well the Freundlich model (R² > 0.97). K_f constants varied between 93 ± 3.1 and 2045 ± 157 for glyphosate and between 99 ± 4.1 and 1517 ± 56 (μg^1‐1/n mL^1/n g^–1) for aminomethylphosphonic acid, showing the broad range of applicability of the proposed method.     

Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry

We have developed a method using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) and isotope dilution quantification to measure atrazine and seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine, diaminochloroatrazine, desisopropylatrazine, desethylatrazine, desethylatrazine mercapturate, atrazine mercaturate and atrazine itself. Our method has good precision (relative standard deviations ranging from 4 to 20% at 5, 10 and 50 ng/mL), extraction efficiencies of 67 to 102% at 5 and 25 ng/mL, relative recoveries of 87 to 112% at 5, 25, 50 and 100 ng/mL limits of detection (LOD) ranging from 0.03 to 2.80 ng/mL. The linear range of our method spans from the analyte LOD to 100 ng/mL (40 ng/mL for atrazine and atrazine mercapturate) with R ² values of greater than 0.999 and errors about the slope of less than 3%. Our method is rapid, cost-effective and suitable for large-scale sample analyses and is easily adaptable to other biological matrices. More importantly, this method will allow us to better assess human exposure to atrazine-related chemicals. Figure A schematic representation showing the elution of the analytes from the solid-phase extraction cartridge onto the analytical column for chromatographic separation prior to MS/MS analysis     

Simultaneous determination of benzotriazole and benzothiazole derivatives in aqueous matrices by mixed-mode solid-phase extraction followed by liquid chromatography–tandem mass spectrometry

An improved selectivity method for the simultaneous determination of four benzotriazoles (benzotriazole, 4-methylbenzotriazole, 5-methylbenzotriazole, and 5,6-dimethyl-1H-benzotriazole) and six benzothiazoles (benzothiazole, 2-hydroxybenzothiazole, 2-benzothiazolamine, mercaptobenzothiazole, 2-methylbenzothiazole, and 2-methylthiobenzothiazole) in aqueous matrices has been developed. Under optimal conditions, analytes are concentrated using a MAX solid-phase extraction (SPE) cartridge, based on divinylbenzene-N-vinylpyrrolidone functionalized with quaternary amine groups, which allows reversed-phase interactions in combination with ionic exchange. Selected compounds are recovered with methanol–acetone 7:3 (v/v) whereas acidic interferences remained attached to the sorbent, and as determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), LOQs for surface, urban and industrial wastewater are in the range of 0.002–0.29 ng/mL. Figures of merit of the method revealed good precision (RSD% <12%), linearity (R ² > 0.99) and accuracy (%R = 80–100%) for surface waters and effluents allowing direct external standard quantification. For more complex samples, such as urban and industrial raw wastewater, either the standard addition method or pseudo-external standard calibration using matrix matched standards are recommended. Analysis of different real samples, surface, urban wastewater and, for the first time, metal industry wastewater, reflected concentrations up to 310 ng/mL. The methylbenzotriazole isomers ratio was also determined.     

Quantification of rosiglitazone in rat plasma and tissues via LC–MS/MS: Method development,validation, and application in pharmacokinetic and tissue distribution studies

A bioanalytical method for the quantification of rosiglitazone in rat plasma and tissues (adipose tissue, heart, brain, bone, and kidney) using LC–MS/MS was developed and validated. Chromatographic separation was achieved on a Gemini C₁₈ column (50 × 4.6 mm, 3 μm) using a mobile phase consisting of 10 mM ammonium formate (pH 4.0) and acetonitrile (10:90, v/v) at a flow rate of 0.8 mL/min and injection volume of 10 μL (internal standard: pioglitazone). LC–MS detection was performed with multiple reaction monitoring mode using target ions at m/z → 358.0 and m/z → 357.67 for rosiglitazone and pioglitazone (internal standard), respectively. The calibration curve showed a good correlation coefficient (r²) over the concentration range of 1–10,000 ng/mL. The mean percentage recoveries of rosiglitazone were found to be over the range of 92.54–96.64%, with detection and lower quantification limit of 0.6 and 1.0 ng/mL, respectively. The developed method was validated per U.S. Food and Drug Administration guidelines and successfully utilized to measure rosiglitazone in plasma and tissue samples. Further, the developed method can be utilized for validating specific organ-targeting delivery systems of rosiglitazone in addition to conventional dosage forms.     

Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study

A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 µm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]⁺ m/z 483.3 for heteroclitin D and [M + H]⁺ m/z 629.3 for the IS. The standard curve was linear (R² ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back‐calculated accuracy and precision. The intra‐ and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacokinetic studies of soyalkaloid A from Portulaca oleracea L. using ultra high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry and its antioxidant activity

Soyalkaloid A was isolated from Portulaca oleracea L. for the first time in our laboratory and then a rapid and sensitive ultra‐high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry (UHPLC–ESI–Q–TOF/MS) method with hesperidin as internal standard (IS) was developed and validated to investigate the pharmacokinetics of soyalkaloid A in rats after oral and intravenous administrations. The analysis was achieved on an Agilent Zorbax Eclipse Plus C₁₈ Column (2.1 × 50 mm, 1.8 μm) by elution with acetonitrile and water (containing 0.1% formic acid), at a flow rate of 0.3 mL/min. The MS analysis was performed in the positive ion mode with monitored ion m/z 227.0814 [M + H]⁺ and 611.1971 [M + H]⁺ for soyalkaloid A and IS, respectively. The linear range was established over the concentration range 7.5–6000 ng/mL (r = 0.9951). The intra‐ and inter‐assay accuracy and precision were between ?4.86‐4.49 and 1.93–9.66, respectively. The lower limits of detection and quantitation observed were 2.1 and 7.4 ng/mL, respectively. The rapid, sensitive and specific UHPLC–ESI–Q–TOF/MS method was successfully applied to a pharmacokinetic study of soyalkaloid A. Moreover, its antioxidant was studied via a 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging assay, the IC₅₀ value being 20.73 ± 0.51 μM.     

Determination of perfluorinated compounds in mollusks by matrix solid-phase dispersion and liquid chromatography–tandem mass spectrometry

Perfluorinated compounds (PFCs) have been used for over 40 years in different commercial and industrial applications mainly as surfactants and surface protectors and have become an important class of marine emerging pollutants. This study presents the development and validation of a new analytical method to determine the simultaneous presence of eight PFCs in different kinds of mollusks using matrix solid-phase dispersion (MSPD) followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Simplicity of the analytical procedure, low volume of solvent and quantity of sample required, low global price, and integration of extraction and clean-up into a single step, are the most important advantages of the developed methodology. Solvent, solid support (dispersing agent), clean-up sorbent, and their amounts were optimized by means of an experimental design. In the final method, 0.5 g of sample are dispersed with 0.2 g of diatomaceous earth and transferred into a polypropylene syringe containing 4 g of silica as clean-up sorbent. Then, analytes are eluted with 20 mL of acetonitrile. The extract is finally concentrated to a final volume of 0.5 mL in methanol, avoiding extract dryness in order to prevent evaporation losses and injected in the LC-MS/MS. The combination of this MSPD protocol with LC-MS/MS afforded detection limits from 0.05 to 0.3 ng g⁻¹. Also, a good linearity was established for the eight PFCs in the range from limit of quantification (LOQ) to 500 ng mL⁻¹ with R ² > 0.9917. The recovery of the method was studied with three types of spiked mollusk and was in the 64–126% range. Moreover, a mussel sample was spiked and aged for more than 1 month and analyzed by the developed method and a reference method, ion-pair extraction, for comparison, producing both methods statistically equal concentration values. The method was finally applied to the determination of PFCs in different kinds of mollusks revealing concentrations up to 8.3 ng g⁻¹ for perfluoroundecanoic acid.     

Simultaneous analysis of ten low‐molecular‐mass organic acids in the tricarboxylic acid cycle and photorespiration pathway in Thalassiosira pseudonana at different growth stages

A method using high‐performance liquid chromatography coupled with tandem mass spectrometry was developed for the simultaneous determination of organic acids in microalgae. o‐Benzylhydroxylamine was used to derivatize the analytes, and stable isotope‐labeled compounds were used as internal standards for precise quantification. The proposed method was evaluated in terms of linearity, recovery, matrix effect, sensitivity, and precision. Linear calibration curves with correlation coefficients >0.99 were obtained over the concentration range of 0.4–40 ng/mL for glycolic acid, 0.1–10 ng/mL for malic acid and oxaloacetic acid, 0.02–2 ng/mL for succinic acid and glyoxylic acid, 4–400 ng/mL for fumaric acid, 20–2000 ng/mL for isocitric acid, 2–200 ng mL⁻¹ for citric acid, 100–10000 ng mL⁻¹ for cis‐aconitic acid, and 1–100 ng mL⁻¹ for α‐ketoglutaric acid. Analyte recoveries were between 80.2 and 115.1%, and the matrix effect was minimal. Low limits of detection (0.003–1 ng/mL) and limits of quantification (0.01–5 ng/mL) were obtained except cis‐aconitic acid. Variations in reproducibility for standard solution at three different concentrations levels were <9%. This is the first report of the simultaneous analysis of ten organic acids in microalgae, which promotes better understanding of their growth state and provides reference value for high‐yield microalgae cultures.     

液相色谱-串联质谱法检测血清中15种甾体激素的动态变化

建立了用液相色谱-串联质谱检测血清中15种甾体激素的方法。在血清样品中加入含内标的乙腈沉淀蛋白质后,用100 mmol/L盐酸羟胺衍生,经Agilent-C18柱(50 mm×3.0 mm,2.7μm)分离,内标法定量。质谱分析采用电喷雾电离(ESI)正离子扫描,多反应监测(MRM)模式检测。数据显示,13种激素在0.05~20 ng/mL范围内线性关系良好(相关系数(R2)不小于0.995 6),检出限不大于1 ng/mL;皮质醇及脱氢表雄酮硫酸酯在50~2 000ng/mL范围内线性关系良好(R2不小于0.997 9),检出限不大于0.5 ng/mL。将该方法应用于女性月经周期激素变化规律的研究,结果表明,15种甾体激素在一个完整的月经周期内有不同特征的动态变化,其中雄激素在滤泡期的后期达到最高值,孕(雌)激素在黄体期达到最高值,而皮质激素则波动不明显。该方法灵敏度高、重复性好,为临床诊疗提供了参考。     

Densitometric high-performance thin-layer chromatography methods for the quantification of oleuropein in Olea europaea leaves and pharmaceutical preparation utilizing normal- and reversed-phase silica gel plates

Two validated, simple and precise densitometric high-performance thin-layer chromatography (HPTLC) quantification methods were proposed for both qualitative and quantitative estimation of oleuropein in Olea europaea leaves and a pharmaceutical product utilizing normal-phase and reversed-phase silica gel TLC plates. In method I, 10 × 20 cm glass plates coated with 0.2 mm thin layers of normal-phase silica gel 60 containing F₂₅₄ (E-Merck, Germany) and a mixture of ethyl acetate‒methanol‒water (8:1:0.5, V/V) were used as the stationary and the mobile phase, respectively. Method II utilized 10 × 20 cm glass-backed plates supporting 0.2 mm layers of RP-18 silica gel 60 containing F₂₅₄ (E-Merck, Germany) as the stationary phase and green solvents mixture composed of ethanol‒water (5.5:4.5, V/V) as the mobile phase. The two methods resulted in sharp, symmetrical, well-resolved peaks at R_F values of 0.47 ± 0.02 and 0.78 ± 0.03 with linearity ranges 200‒1400 ng/spot (r² = 0.9994) and 200‒1400 ng/spot (r² = 0.9996) for method I and method II, respectively. Spots corresponding to oleuropein were scanned at 200 nm. The two methods complied with the ICH guidelines for validation. Due to simplicity, low cost and short analysis time, the methods can be good alternatives for the quality control of different products containing olive leaves extract or pure oleuropein.

     

l‐Cysteine‐modified magnetic microspheres for extraction and quantification of saxitoxin in rat plasma with liquid chromatography and tandem mass spectrometry

Saxitoxin, which is one of the most typical paralytic shellfish poisoning toxins, ranks the highest intoxication rate of marine biological poisoning cases globally. Efficient clean‐up and extraction of saxitoxin from complex biological matrices are imperative for the analysis and concentration monitoring of the toxin when correlative poisoning cases happen. Herein, l ‐cysteine‐modified magnetic microspheres based on metal‐organic coordination were synthesized by a facile approach and applied for magnetic solid‐phase extraction of saxitoxin from rat plasma samples before liquid chromatography–tandem mass spectrometry detection. Parameters, including adsorbent amount, extraction time, desorption solution, and desorption time that could affect the extraction efficiency, were respectively investigated. The developed method demonstrated good linearity in the range of 5–300 ng/mL (R² = 0.9985) with a limit of quantification of 5 ng/mL and a limit of detection of 0.5 ng/mL, acceptable accuracy. and precision of within‐run and between‐run.