Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma.

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe3O4) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe3O4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R2 > 0.9987) over the range (0.001-5.0 µg/mL), limit of detection (0.003 µg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

Correlation analysis based on the hydropathy properties of non-steroidal anti-inflammatory drugs in solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography (HPLC) with photodiode array detection and their applications to biological samples.

In the present work we analyzed the hydrophobicity and hydrophilicity properties of several non-steroidal anti-inflammatory drugs (NSAIDs) by investigating the structural changes of the dynamic hydrogen bond network in order to predict the extraction recovery of NSAIDs from biological fluids set by solid phase extraction (SPE). This work allows investigating the relationship between theoretical descriptors and experimental data using a parameter free method with a strong correlation (Pearson correlation 0.95, p-value 0.0003). The identification and quantification of analytes in human plasma were carried out by high performance liquid chromatography coupled with photodiode array detection (HPLC-PDA) using a Kinetex Evo C18 (150 x 4.6 mm I.D) protected by a guard column and a mixture of acetonitrile and 10 mM phosphate buffer (pH 2.5) (50:50, v/v) as mobile phase at isocratic conditions. Accuracy (BIAS%) ranged within -2.33% and + 8.05% while precision (RSD%) was less than 5.73%.The mean extraction recovery of the carprofen (IS) was 84.1% and the recovery of NSAIDs from human plasma ranged between 81.9% to 86.6%. LODs and LOQs for all the investigated NSAIDs were 0.003 and 0.01 µg/mL, respectively. The method was validated according to the ICH guide line in the range 0.010-20.0 µg/mL.

Meropenem and ciprofloxacin in complicated gastric surgery for cancer patients: A simple SPE-UHPLC-PDA method for their determination in human plasma.

A simple and rapid ultra-high-performance liquid chromatographic (UHPLC) for the simultaneous determination of meropenem and ciprofloxacin in human plasma was developed and validated. All of the analytes were separated in <5 min. A solid-phase extraction method was applied from sample preparation. Analytical separation was performed on a Poroshell SB C18 column (50 × 2.1 mm, 2.7 µm particle size) with photodiode array (PDA) detection. Meropenem and ciprofloxacin were determined at wavelengths of 300 and 277 nm, respectively. The mobile phase was a mixture of acetonitrile-10 mm ammonium acetate-methanol in gradient elution. The method has been validated for both drugs in gastric surgery for cancer patients. The method showed good linearity with correlation coefficients, r2  = 0.994 for the two drugs, as well as high precision (RSD < 10.5% in each case); accuracy ranged from -5.8 to +6.0%. The limit of quantitation of the two drugs was established at 0.02 and 0.01 µg/mL, respectively. Meropenem, ciprofloxacin and the internal standard were extracted from human plasma with a mean recovery ranging from 92.5 to 98.6%. The method was applied to quantify the drugs dosage in complicated gastric surgery patients.

Dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of NSAIDs in human plasma and urine.

A novel, rapid, simple graphene/Fe3O4 based dispersive magnetic solid phase extraction was developed for the simultaneous separation/preconcentration and determination of non steroidal anti-inflammatory drugs with ultra high performance liquid chromatography coupled with photodiode array detection. Several parameters influencing the extraction efficiency of the investigated analytes such as the extraction time, the amount of graphene/Fe3O4, the sample pH, the ionic strength and the elution solvent were evaluated and optimized. Under optimal conditions, the linearity was in the range of 0.002-25 µg/mL for furprofen, diclofenac and ketoprofen, 0.003-25 for flurbiprofen, naproxen and fenbufen, 0.004-25 for indoprofen with a good coefficient of determination (R2> 0.9991) for each analyte. The inter-and- intra day accuracy (BIAS%) for human plasma and urine ranged between -7.15% to 6.20% and -5.17% to 4.87%, respectively.The precision (RSD%) in human plasma and urine was less than 8.67% and 8.92%, respectively. The proposed method was applied to the determination of NSAIDs in human plasma and urine.

Air assisted dispersive liquid-liquid microextraction with solidification of the floating organic droplets (AA-DLLME-SFO) and UHPLC-PDA method: Application to antibiotics analysis in human plasma of hospital acquired pneumonia patients.

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of metronidazole, meropenem, ciprofloxacin, linezolid and piperacillin in human plasma and applied to patients suffering from hospital acquired pneumonia (HAP). The method uses an air assisted dispersive liquid-liquid microextraction for sample preparation. All parameters in the extraction step, including selection of extractant, amount of extractant, ionic strength, pH, and extraction cycles, were investigated and optimized. Chromatography was carried out using a Poroshell 120 SB C18 (50 × 2.1 mm I.D. 2.6 µm particle size) column and a gradient mobile phase consisting of ammonium acetate buffer (10 mM, pH 4.0) (eluent A); and a mixture of acetonitrile-methanol in a ratio (80/20)(eluent B). Ulifloxacin was used as internal standard. The method demonstrated good linearity with correlation coefficients, r2 > 0.9995 for the drugs, as well as high precision (RSD% ≤ 9.87%), accuracy ranged from -8.14% to +8.98. The enrichment factor (EF) obtained ranged within 87 and 121. During the validation, the concentrations of the analytes were found to be stable after 3 freeze-thaw cycles and for at least 24 h after extraction. Subsequently, this method was used to quantify the drugs in patients with HAP in order to establish if the dosage regimen given was sufficient to eradicate the infection at the target site.

A green deep eutectic solvent dispersive liquid-liquid micro-extraction (DES-DLLME) for the UHPLC-PDA determination of oxyprenylated phenylpropanoids in olive, soy, peanuts, corn, and sunflower oil.

A green dispersive liquid-liquid microextraction (DLLME) using deep eutectic solvent (DES) as the extracting solvent has been developed and applied for the simultaneous quantification of ferulic acid, umbelliferone, boropinic acid, 7-isopentenyloxycoumarin, 4'-geranyloxyferulic acid (GOFA), and auraptene in some vegetable oils using ultra high performance liquid chromatography (UHPLC) with photodiode array detection (PDA). All parameters in the extraction step, including selection and loading of both extracting and dispersing solvents, amount of both extractant and disperser solvent were investigated and optimized. PhAA/TMG DES achieved higher recovery and enrichment factor compared to other DESs. The validated method showed good linearity with correlation coefficients, r2>0.9990 for all the analytes. Furthermore, this is the first time that eco-friendly solvents are used for the extraction of oxyprenylated phenylpropanoids and the corresponding extract analyzed with ultra high performance liquid chromatography with photodiode array detection.

Meropenem, levofloxacin and linezolid in human plasma of critical care patients: A fast semi-automated micro-extraction by packed sorbent UHPLC-PDA method for their simultaneous determination.

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of meropenem, linezolid, and levofloxacin in human plasma and applied in human plasma of critical care patients. A semi-automated microextraction by packed sorbent (MEPS) for sample preparation was used. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration - ejection cycles) and in the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of elution solvent through the device) were statistically significant when the recovery was used as response. The method showed good linearity with correlation coefficients, r2>0.9991 for the three drugs, as well as high precision (RSD%<10.83% in each case). Accuracy ranged from -7.8% to +6.7%. The limit of quantification of the three drugs was established at 0.01µg/mL for linezolid and levofloxacin and 0.02µg/mL for meropenem. Linezolid, meropenem, levofloxacin and the internal standard were extracted from human plasma with a mean recovery ranged from 92.4% to 97.4%. During validation, the concentration of meropenem, linezolid and levofloxacin was found to be stable after 3 freeze-thaw cycles and for at least 24h after extraction. This method will be subsequently used to quantify the drugs in patients to establish if the dosage regimen given is sufficient to eradicate the infection at the target site.

Development of a dried blood spot HPLC-PDA method for the analysis of linezolid and ciprofloxacin in hospital-acquired pneumonia patients.

This study developed a high performance liquid chromatography (HPLC) method involving dried blood spotting (DBS) as a sampling method for the therapeutic drug monitoring of antimicrobial combination therapy with linezolid and ciprofloxacin. DBS for standards, quality control samples, and patient samples was excised and then extracted using a mixture of methanol/water/formic acid 80:20:0.1 (v/v/v), respectively. Linezolid (LZD) and ciprofloxacin (CPR) were measured by HPLC using a Kinetex EVO C18 (100 × 4.6 mm I.D. 2.6 µm particle size). Mobile phase consisted of 10 mM ammonium acetate (A) and a mixture of acetonitrile and methanol (B), both containing 0.1% triethylamine, in gradient elution. Detection was carried out at 251 nm for linezolid (LZD) and 277 for ciprofloxacin (CPR). Ulifloxacin was used as an internal standard. The internal standard, LZD, and CPR were eluted in 7.90, 7.18, and 8.89 min, respectively. Total run-time was 20 min. Calibration curves were constructed in the range of 0.05-30 µg/mL for LZD and 0.02-10 µg/mL for CPR, respectively. The intra- and inter-day precision (RSD values) did not exceed 9.43%, the intra- and inter-day accuracy (accuracy %) ranged between 96.2 and 106.2%. Haematocrit (Hct) effects were investigated to obtain a linear correlation between haematocrit values and volume of blood. Copyright © 2017 John Wiley & Sons, Ltd.

Development and validation of a fast micro-extraction by packed sorbent UHPLC-PDA method for the simultaneous determination of linezolid and ciprofloxacin in human plasma from patients with hospital-acquired pneumonia.

An ultra high-performance liquid chromatographic (UHPLC) method with PDA detection was developed and validated for the simultaneous quantification of linezolid and ciprofloxacin in human plasma and applied in hospital acquired pneumonia patients (HAP). The method uses a semi-automated microextraction by packed sorbent for sample preparation. All parameters in the extraction step (pH, sample volume, sample dilution and number of aspiration - ejection cycles) and in the desorption step (percentage of acetonitrile in the solvent of elution and number of aspirations of elution solvent through the device) were statistically significant when the recovery was used as response. The method showed good linearity with correlation coefficients, r2>0.9995 for the two drugs, as well as high precision (RSD%<9.77% in each case), accuracy ranged from -6.2% to +8.2. The limit of quantification of the two drugs was established at 0.01 and 0.02µg/mL for ciprofloxacin and linezolid, respectively. Linezolid, ciprofloxacin and internal standard were extracted from human plasma with a mean recovery ranging from 92.4% to 97.4%. During validation, the concentrations of linezolid and ciprofloxacin were found to be stable after 3 freeze-thaw cycles and for at least 24h after extraction. This method will subsequently be used to quantify the drugs dosage in patients with HAP to establish if the dosage regimen given is sufficient to eradicate the infection at the target site.

Development and validation of a MEPS-UHPLC-PDA method for determination of ulifloxacin in human plasma and urine of patients with peripheral arterial disease.

A novel sensitive analytical method based on the use of a semi-automatic microextraction by packed sorbents (MEPS) techniques combined with ultra high-performance liquid chromatography (UHPLC) with PDA detection has been developed and validate for the analysis of ulifloxacin, the active metabolite of prulifloxacin using danofloxacin as internal standard in human plasma and urine. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with a Waters BEH C18 (50×2.1mm I.D., 1.7µm particle size) analytical column, a mixture of 10mM ammonium acetate (pH 3.0) (A) with and acetonitrile (B) both containing 1% triethylamine were used as mobile phase, at a flow rate of 0.6mL/min in gradient elution, and detection wavelength of 272nm. This method is linear in concentration range of 0.02-10.0µg/mL for plasma and urine, respectively. The limit of quantitation was 20ng/mL for the two fluids. The recoveries of the method were 95% for ulifloxacin in human plasma and urine and 95.5% for the internal standard. Intra- and inter- assay precision and accuracy for ulifloxacin were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma and urine concentrations of ulifloxacin in patients suffering from Peripheral Arterial Disease and for pharmacokinetics study.

Optimisation by response surface methodology of microextraction by packed sorbent of non steroidal anti-inflammatory drugs and ultra-high performance liquid chromatography analysis of dialyzed samples.

A procedure based on microextraction by packed sorbent (MEPS) followed by ultra-high performance liquid chromatography (UHPLC) with photodiode array (PDA) detection has been developed for the analysis of seven selected non steroidal anti-inflammatory drugs (NSAIDs) in human dialysates. The influence on MEPS efficiency of pH of the sample, pH of the washing solvent and methanol content in the hydro-alcoholic elution mixture has been investigated by response surface methodology based on a Box-Behnken design of experiments. Among the above factors, pH of sample is the variable that mostly influences MEPS recovery. UHPLC separation of the NSAIDs was completed within less than 4min under isocratic elution conditions on a Fortis SpeedCore C18 column (150×4.6mm I.D., 2.6µm) using acetonitrile-phosphate buffer as the mobile phase. Calibration curves of the NSAIDs were linear over the concentration range 0.025-15µg/mL, with correlation coefficients≥0.998. Intra- and inter-assay relative standard deviations were <8% and recovery values ranged from 94% to 100% for the quality control samples. The results reveal that the developed MEPS/PDA-UHPLC method exhibits a good accuracy and precision and is well suited for the rapid analysis of human dialysate from patients treated with the selected NSAIDs.

Bioanalytical method development for quantification of ulifloxacin, fenbufen and felbinac in rat plasma by solid-phase extraction (SPE) and HPLC with PDA detection.

A procedure based on solid-phase extraction (SPE) followed by high performance liquid chromatography (HPLC) with PDA detection has been developed for the analysis of multiple drugs in rat plasma. The analytes evaluated were ulifloxacin, fenbufen and felbinac. Eight different solid phase extraction cartridges were tested to evaluate their applicability for the isolation of drugs from rat plasma. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by HPLC using a Kinetex C18 EVO column and acetonitrile-10mM ammonium acetate-methanol as the mobile phase under gradient elution conditions. SPE combined with HPLC-PDA allowed the determination of drugs over a linear range of 0.05-15 µg/mL for ulifloxacin while 0.5-50 µg/mL for felbinac and fenbufen, with limit of detection at 0.05 for ulifloxacin and 0.5 for felbinac and fenbufen. Bond Elut Plexa sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 93.54% with relative standard deviation (RSD) <10%. The method was applied with good accuracy and precision in the determination of ulifloxacin, fenbufen and felbinac in rat plasma obtained from rats treated with selected drugs. This method permits its application to pharmacokinetic and pharmacodynamic studies of these analytes and will facilitate detailed investigations on the interactions between new fluoroquinolones and fenbufen.

A Novel Biological Role of α-Mangostin in Modulating Inflammatory Response Through the Activation of SIRT-1 Signaling Pathway.

Several studies have shown that xanthones obtained from Garcinia Mangostana (GM) have remarkable biological activities. α-mangostin (α-MG) is the main constituent of the fruit hull of the GM. Several findings have suggested that SIRT-1, a nuclear histone deacetylase, could influence cellular function by the inhibition of NF-kB signaling. ROS can inhibit SIRT-1 activity by initiating oxidative modifications on its cysteine residues, and suppression of SIRT-1 enhances the NF-κB signaling resulting in inflammatory responses. The goals of the present study were to evaluate the quantity of α-MG in the methanolic extract of GM (Vithagroup Spa) and to investigate the activity of this xanthone in U937 cell line and in human monocytes from responsive to inflammatory insult analyzing the possible changes on the activation of SIRT-1 protein via NF-Kb. Cells were treated with the methanolic extract of GM and/or LPS. The chromatographic separation of α-MG was performed by an HPLC analysis. EX 527, a specific SIRT-1 inhibitor, was used to determine if SIRT-1/NfkB signaling pathway might be involved in α-MG action on cells. Our results show that α-MG inhibits p65 acetylation and down-regulates the pro-inflammatory gene products as COX-2, iNOS via SIRT-1 activation. Cells treated with EX 527 showed an up-regulation of NFkB acetylation and an over expression of inducible enzymes and their product of catalysis (NO and PGE2). These results suggest that α-MG may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process. J. Cell. Physiol. 231: 2439-2451, 2016. © 2016 Wiley Periodicals, Inc.

Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.

A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 µg/mL for octreotide and 0.2 µg/mL for gabexate mesylate metabolite, and matrix matched standard curves showed a good linearity up to 15 µg/mL. In the entire analytical range the intra- and inter-day precision (RSD%) values were respectively ≤5.9% and ≤3.1% for octreotide and ≤2.0% and ≤3.9% for gabexate mesylate metabolite. For both analytes the intra- and inter-day accuracy (bias) values ranged respectively from -6.8 to -2.5% and from -4.6 to -5.7%. This method utilizes derivatization with NBD-F and provides adequate sensitivity for both drugs.

Analysis of anti-inflammatory enantiomers by HPLC in human plasma and urine: a review.

The analysis of drugs in various biological fluids is an important criterion for the determination of the physiological performance of a drug. NSAIDs are non-selective inhibitors of prostaglandin biosynthesis and indicated for the acute or long-term treatment of the signs and symptoms of rheumatoid arthritis and osteoarthritis. This paper reviews the recent developments in bioanalysis of these drugs. In particular, sample preparation end, handling procedures, chromatographic conditions and detection methods are discussed. A summary of published HPLC assays for individual antiinflammatory drugs is included.