Precipitation Of Plasma Proteins Research Articles

Determination and pharmacokinetics of DT-13 in rat plasma by LC–MS

A sensitive and specific LC–MS assay for DT-13 in rat plasma was developed. DT-13 is an active steroidal saponin present in Liriopes Radix and is developed as an anti-tumor drug candidate. The samples were extracted by acetonitrile-mediated plasma protein precipitation. The chromatographic separation was carried out using a Ultimate C18 column (250mm×4.6mm, i.d., 5μm) with a mobile phase composed of acetonitrile: 5mmol/L aqueous ammonium acetate (60:40, v:v). The method was validated and the specificity, linearity (r2=0.9980 within 10–1000ng/mL), lower limit of quantitation (LLOQ, 10ng/mL), precision (intra- and inter-day <12.3%), accuracy (93.4–106.3%), recovery (91.0±4.7%) and stability were determined. The method was applied to the pharmacokinetic study of DT-13 in rat plasma after intravenous and intragastric administration. The results showed DT-13 underwent a prolonged absorption and slow elimination with a low oral bioavailability (5.51%) in rats.

Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin, lomefloxacin and ciprofloxacin in human plasma

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC-FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single-step precipitation of plasma proteins followed by vortex-mixing and centrifugation. Chromatographic separation was achieved within 7 min on a reversed-phase C(18) column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)-methanol (82:18, v/v) pumped isocratically at 1.2 mL/min. The detector was set at excitation/emission wavelengths of 278/450 nm. Calibration curves were linear (r(2) ≥ 0.994) in the range of 0.02-5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC-FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic-based studies with those antimicrobial agents.

Simultaneous Quantification of Levetiracetam and Gabapentin in Plasma by Ultra-Pressure Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection

Gabapentin (Neurontin) and levetiracetam (Keppra) are anticonvulsants with novel structures and suggested therapeutic ranges of 2-10 mg/L and 6-20 mg/L, respectively. Gabapentin is also used extensively to manage neuropathic pain, and for this indication, wherein higher doses are prescribed, plasma concentrations of 15-30 mg/L are typical. Here, we describe a simple rapid assay to support therapeutic drug monitoring of gabapentin and levetiracetam in plasma by ultra-pressure liquid chromatography couples to tandem mass spectrometry (UPLC-MS/MS) detection. After the addition of internal standard and protein precipitation of patient plasma with methanol:acetonitrile in a 50:50 ratio, 1 μL of supernatant sample is injected onto an Acquity UPLC HSS T3, 1.8 μm, 2.1 × 50 mm (Waters) column. Elution occurs using a linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into a Waters Acquity UPLC TQD, operating in a positive mode to detect gabapentin at transition 172.18 > 154.11, levetiracetam at 171.11 > 126, and internal standard (3-amino-2-naphthoic acid) at 188.06 > 170. Secondary transitions for each analyte are also monitored for gabapentin at 172.18 > 137.06, levetiracetam at 171.11 > 154, and internal standard at 188.06 > 115. Runtime is 1.5 minutes per injection with baseline resolved chromatographic separation. The analytical measurement ranges were 1-150 mg/L for gabapentin and for levetiracetam. Intra-assay imprecision by the coefficient of variance (CV) was less than 8% and interassay CV was less than 5% for both analytes, at 4 different concentrations. Results obtained from patient samples were compared with results generated by established high-performance liquid chromatography-UV methods with the following regression statistics: y = 1.12x - 0.77, r = 0.996, Sy, x = 0.89, and n = 29 for gabapentin and y = 0.991x + 0.70, r = 0.997, Sy, x = 2.24, and n = 30 for levetiracetam. No analytical interferences were identified. : In summary, a simple reliable UPLC-MS/MS method was developed and validated for routine clinical monitoring of gabapentin and levetiracetam.

High‐performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization

In this study, we investigated a simple, sensitive and reliable liquid chromatography-fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). For the first time, FMOC-Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC-Cl to form memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions, which can be very compatible for LC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C(18) column (DIAMONSIL 150 × 4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025-5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 4.46-12.19 and 5.23-11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.

A strategy for liquid chromatography/tandem mass spectrometry based quantitation of pegylated protein drugs in plasma using plasma protein precipitation with water‐miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection

Recently, we have developed liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods for the quantitation of pegylated therapeutic proteins in plasma. The methods are based on the LC/MS/MS detection of a surrogate peptide generated from trypsin digestion of the therapeutic protein. Various parameters related to the bioanalytical methods were evaluated and optimized, including the preparation of calibration standards and quality control samples, sample extraction, internal standard selection and its stage of addition, trypsin digestion, and non-specific binding. In this paper, we report the development of a method for a specific pegylated therapeutic protein and detail the various optimization steps undertaken. Simple extraction of the pegylated therapeutic protein from plasma was achieved via the precipitation of the endogenous proteins in plasma using acidic isopropanol and the resulting supernatant extract was subjected to trypsin digestion. A unique tryptic peptide arising from the pegylated therapeutic protein was used for LC/MS/MS-based detection and quantitation. A protein and a peptide were used as internal standards, with the former added before the sample extraction and the latter after the sample extraction. The method developed is simple, sensitive, specific and rugged, and has been implemented in a high throughput 96-well format to analyze plasma samples from in vivo studies. A required lower limit of quantitation (LLOQ) of 10 ng/mL, expressed in terms of the concentration of the protein drug, was easily achieved.

Development and Validation of a High-Performance Liquid Chromatography Method for Determination of Cefquinome Concentrations in Sheep Plasma and Its Application to Pharmacokinetic Studies

Cefquinome has a broad spectrum of antibacterial activity and was developed especially for use in animals. A simple and sensitive high-performance liquid chromatography (HPLC) method with UV-visible detection for quantification of cefquinome concentrations in sheep plasma was developed and validated. Separation of cefquinome from plasma components was achieved on a Phenomenex Gemini C(18) column (250 mm by 4.6 mm; internal diameter [i.d.], 5 μm). The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid in water and was delivered at a rate of 0.9 ml/min. A simple and rapid sample preparation involved the addition of methanol to 200 μl of plasma to precipitate plasma proteins followed by direct injection of 50 μl of supernatant into the high-performance liquid chromatography system. The linearity range of the proposed method was 0.02 to 12 μg/ml. The intraday and interday coefficients of variation obtained from cefquinome were less than 5%, and biases ranged from -3.76% to 1.24%. Mean recovery based on low-, medium-, and high-quality control standards ranged between 92.0 and 93.9%. Plasma samples were found to be stable in various storage conditions (freeze-thaw, postpreparative, short-term, and long-term stability). The method described was found to be readily available, practicable, cheap, rapid, sensitive, precise, and accurate. It was successfully applied to the study of the pharmacokinetics of cefquinome in sheep. This method can be very useful and an alternate to performing pharmacokinetic studies in the determination of cefquinome for clinical use.

Confirmatory Analysis of Continuous Erythropoietin Receptor Activator and Erythropoietin Analogues in Equine Plasma by LC−MS for Doping Control

Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.

Quantitative dynamic nuclear polarization‐NMR on blood plasma for assays of drug metabolism

Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.

Determination of Linezolid in Human Plasma by High-Performance Liquid Chromatography With Ultraviolet Detection

A high-performance liquid chromatographic method for the determination of linezolid in human plasma was developed and validated. After precipitation of plasma proteins with perchloric acid, the protein-free supernatant was separated by isocratic reverse-phase chromatography on a X Bridge C18 column. The mobile phase consisted of a mixture of phosphoric acid 0.05%: acetonitrile (75:25, v/v) with a flow rate of 1 mL/min. The column elute was monitored at 254 nm. The method was linear from 0.2 to 48 mg/L (mean r2 = 0.9996, n = 10). The observed intra- and inter-day assay imprecision ranged from 2.83% to 8.16% (18.80% at the lower limit of quantification); inaccuracy varied between -0.33% and 8.18%. Mean drug recovery was 99.8% for linezolid and 90.0% for the internal standard (para-toluic acid). The method was found to be precise and accurate and suitable for therapeutic drug monitoring of linezolid in routine clinical practice.

The Application of Trichloroacetic Acid as an Ion Pairing Reagent in LC–MS–MS Method Development for Highly Polar Aminoglycoside Compounds

A simple, sensitive and robust liquid chromatography-tandem mass spectrometry method was developed and validated for highly polar aminoglycoside compounds gentamicin, kanamycin and apramycin. The effect of trichloroacetic acid (TCA) concentration on plasma protein precipitation and sample recovery was studied and an optimized concentration of 25–30% TCA were determined that gives the best sample recovery for aminoglycosides from rat plasma. The effect of TCA concentration on the chromatographic behavior of gentamicin and tobramycin was studied on a Synergy Max RP-column using a mobile phase with a pH of 2.78. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 1–5,000, 20–10,000, 10–10,000 ng mL−1 with precisions of 2.6–4.1, 3.3–5.0, 1.5–9.9%, and accuracies of 94.7–103.7, 87.9–104.9, 91.3–103.6% for gentamicin, kanamycin and apramycin, respectively. The LLOQs corresponding with a coefficient of variation less than 20% were 1, 20 and 10 ng mL−1 for gentamicin, kanamycin and apramycin, respectively.

Analytical issues in HPLC/MS/MS simultaneous assay of furosemide, spironolactone and canrenone in human plasma samples

A new sensitive HPLC/MS/MS method for simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples is presented. Electrospray ionization source (ESI) has been used. The tandem MS detection was performed under MRM conditions, in the negative ion mode for furosemide and indapamide (internal standard 1) and in the positive ion mode for spironolactone, canrenone and nitrazepam (internal standard 2). A simple plasma protein precipitation with acetonitrile was used as sample preparation technique. The chromatographic separation was carried out under the reversed phase mechanism, on a 250 mm length column packed with octadecyl modified silicagel and thermostated at 35 degrees C. The column was operated under isocratic conditions (3:7 aqueous 0.1% formic acid and methanol, v/v) at a flow rate of 0.8 mL/min. Quantitation intervals of 20-1600 ng/mL for furosemide and 2-100 ng/mL for spironolactone and canrenone have been concluded through validation. Precision and accuracy were situated within the accepted thresholds (maximum 15% relative standard deviation and +/-15% percentage bias). The most sensitive aspects relating to the analytical method development and validation were highlighted and critically assessed in order to reach an objective opinion about the real performances and inherent applicability of the method in bioanalysis.

English

&nbsp; Zerumbone (ZER) is a sesquiterpene derived from Zingiber zerumbet smith, family Zingiberaceae. It has been shown to possess anti-cancer and apoptosis-inducing properties against various human tumour cells as well as&nbsp;in vivo&nbsp;against a number of induced malignancies in mice. In this study a&nbsp;simple, specific and accurate high performance liquid chromatographic method for determination of ZER in micro-volumes human plasma (|&nbsp;1.5 ml) was developed and validated. ZER and its analogue &alpha;-Humulene as internal standard were easy recovered by simple one step plasma protein precipitation using acetonitrile and separated in isocratic mobile phase, on reverse phase-C18&nbsp;column. The effluent was monitored by Photodiode Array (PDA) detector and at a flow rate of 1.0 ml/min. The linearity of proposed method was 2 &ndash; 15 &micro;g/ml. The intra-day and inter-day coefficient of variation and percent error values of the method were less than 15% and mean recovery was more than 90% for both ZER and &alpha;-Humulene. This method was found to be precise, specific, accurate and robust for detection and analysis of ZER in human plasma. &nbsp; Key words:&nbsp;ZER, humulene, HPLC, human plasma.

Evaluation and performance of desorption electrospray ionization using a triple quadrupole mass spectrometer for quantitation of pharmaceuticals in plasma

The present work describes the methodology and investigates the performance of desorption electrospray ionization (DESI) combined with a triple quadrupole mass spectrometer for the quantitation of small drug molecules in human plasma. Amoxepine, atenolol, carbamazepine, clozapine, prazosin, propranolol and verapamil were selected as target analytes while terfenadine was selected as the internal standard common to each of the analytes. Protein precipitation of human plasma using acetonitrile was utilized for all samples. Limits of detection were determined for all analytes in plasma and shown to be in the range 0.2-40 ng/mL. Quantitative analysis of amoxepine, prazosin and verapamil was performed over the range 20-7400 ng/mL and shown to be linear in all cases with R(2) >0.99. In most cases, the precision (relative standard deviation) and accuracy (relative error) of each method were less than or equal to 20%, respectively. The performance of the combined techniques made it possible to analyze each sample in 15 s illustrating DESI tandem mass spectrometry (MS/MS) as powerful tool for the quantitation of analytes in deproteinized human plasma.

Simultaneous RP‐HPLC‐DAD quantification of bromocriptine, haloperidol and its diazepane structural analog in rat plasma with droperidol as internal standard for application to drug‐interaction pharmacokinetics

A simple and rapid RP-HPLC-DAD method was developed and validated for simultaneous determination of the dopamine antagonists haloperidol, its diazepane analog, and the dopamine agonist bromocriptine in rat plasma, to perform pharmacokinetic drug-interaction studies. Samples were prepared for analysis by acetonitrile (22.0 microg/mL) plasma protein precipitation with droperidol as an internal standard, followed by a double-step liquid-liquid extraction with hexane : chloroform (70:30) prior to C-18 separation. Isocratic elution was achieved using a 0.1% (v/v) trifluoroacetic acid in deionized water, methanol and acetonitrile (45/27.5/27.5, v/v/v). Triple-wavelength diode-array detection at the lambda(max) of 245 nm for haloperidol, 254 nm for the diazepane analog and droperidol, and 240 nm for bromocriptine was carried out. The LLOQ of DAL, HAL, and BCT were 45.0, 56.1, and 150 ng/mL, respectively. In rats, the estimated pharmacokinetic parameters (i.e., t(1/2), CL, and V(ss)) of HAL when administered with DAL and BCT were t(1/2) = 16.4 min, V(ss) = 0.541 L/kg for HAL, t(1/2) = 28.0 min, V(ss) = 2.00 L/kg for DAL, and t(1/2) = 24.0 min, V(ss) = 0.106 L/kg for BCT. The PK parameters for HAL differed significantly from those previously reported, which may be an indication of a drug-drug interaction.

Turbulent Flow Chromatography TFC-tandem mass spectrometry supporting in vitro/ vivo studies of NCEs in high throughput fashion

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput.

Bioanalysis of pentoxifylline and related metabolites in plasma samples through LC‐MS/MS

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a 'pure' reversed-phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C-18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration-time patterns of these compounds follow typical metabolic profiles. It has been advanced that in-vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches.

Acute Effects of Dietary Glycemic Index on Antioxidant Capacity in a Nutrient‐controlled Feeding Study

Oxidative stress, caused by an imbalance between antioxidant capacity and reactive oxygen species, may be an early event in a metabolic cascade elicited by a high glycemic index (GI) diet, ultimately increasing the risk for cardiovascular disease and diabetes. We conducted a feeding study to evaluate the acute effects of low-GI compared with high-GI diets on oxidative stress and cardiovascular disease risk factors. The crossover study comprised two 10-day in-patient admissions to a clinical research center. For the admissions, 12 overweight or obese (BMI: 27-45 kg/m(2)) male subjects aged 18-35 years consumed low-GI or high-GI diets controlled for potentially confounding nutrients. On day 7, after an overnight fast and then during a 5-h postprandial period, we assessed total antioxidant capacity (total and perchloric acid (PCA) protein-precipitated plasma oxygen radical absorbance capacity (ORAC) assay) and oxidative stress status (urinary F(2alpha)-isoprostanes (F(2)IP)). On day 10, we measured cardiovascular disease risk factors. Under fasting conditions, total antioxidant capacity was significantly higher during the low-GI vs. high-GI diet based on total ORAC (11,736 +/- 668 vs. 10,381 +/- 612 micromol Trolox equivalents/l, P = 0.002) and PCA-ORAC (1,276 +/- 96 vs. 1,210 +/- 96 micromol Trolox equivalents/l, P = 0.02). Area under the postprandial response curve also differed significantly between the two diets for total ORAC and PCA-ORAC. No diet effects were observed for the other variables. Enhancement in plasma total antioxidant capacity occurs within 1 week on a low-GI diet, before changes in other risk factors, raising the possibility that this phenomenon may mediate, at least in part, the previously reported effects of GI on health.

Neutralization of Bothrops mattogrossensis snake venom from Bolivia: Experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation

Polyspecific bothropic/crotalic and bothropic/lachesic antivenoms were produced in Bolivia by immunizing two donkeys with the venoms of Bothrops mattogrossensis and Crotalus durissus terrificus and one llama with the venoms of B. mattogrossensis and Lachesis muta. These antivenoms are currently being used for snakebite envenomation in Bolivia. The rationale for using these animals is that donkeys and llamas are better adapted than horses to the high altitudes in South America and constitute good alternatives for antivenom production in these regions. Plasma was fractionated by caprylic acid precipitation of non-immunoglobulin plasma proteins, to obtain whole IgG preparations. Donkey-derived antivenom showed one band of 150 kDa when analyzed by SDS–PAGE, whereas llama antivenom presented two immunoglobulin bands, of 170 kDa and 120 kDa, the latter corresponding to the heavy-chain antibodies present in camelid sera. The effectiveness of these antivenoms to neutralize lethal, hemorrhagic, myotoxic, edema-forming, and defibrinogenating activities of the venom of B. mattogrossensis from Bolivia, a species formerly known as Bothrops neuwiedii, was assessed at the experimental level. Although llama antivenom has a total protein concentration four times lower than donkey antivenom, both preparations have similar neutralizing capacity against all toxic activities assessed. Llama and donkey IgG-based antivenoms are effective in the neutralization of B. mattogrossensis venom and represent valuable alternatives for antivenom manufacture in highland regions of South America.

A novel strategy for reducing phospholipids-based matrix effect in LC–ESI-MS bioanalysis by means of HybridSPE

A novel strategy to minimize phospholipids-based matrix effects in bioanalytical LC–MS/MS assays was evaluated. The phospholipids-based matrix effect was investigated with a commercially available electrospray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. A systematic comparison approach of two sample preparation procedures was performed. In particular, the matrix effect on mass spectrometry response in rat and human plasma samples was studied by comparing sample extracts obtained by means of a conventional plasma protein precipitation with acetonitrile and the novel HybridSPE-Precipitation procedure. The HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure which combines the simplicity of precipitation with the selectivity of SPE allows to obtain much cleaner extracts than with conventional procedures. The effective targeted removal of phospholipids and proteins in biological plasma samples achieved with the HybridSPE-Precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects.

Determination of free captopril in human plasma by liquid chromatography with mass spectrometry detection

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of captopril after precolumn derivatization with p-bromo-phenacyl-bromide in human plasma was validated. Plasma samples were analysed on a monolithic column (Cromolith Performance-RP 18e, 100 mm × 4.6 mm I.D., 3 μm) under isocratic conditions using a mobile phase of a 40:60 (v/v) mixture of acetonitrile and 0.1% (v/v) formic acid in water. The flow rate was 1 mL/min at the column temperature of 30 °C. In these chromatographic conditions, the retention time was 4.4 min for captopril derivative. The detection of the analyte was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were 216, 253, 255, 268, 270 m/ z derived from 415 m/ z for derivatized captopril. The sample preparation was very simple and consisted in plasma protein precipitation from 0.2 mL plasma using 0.3 mL methanol after the derivatization reaction was completed. Calibration curves were generated over the range of 10–3000 ng/mL with values for coefficient of correlation greater than 0.993 and by using a weighted (1/ y 2) quadratic regression. The values for precision (CV %) and accuracy (relative error %) at quantification limit were less than 9.9% and 3.9%, for within- and between-run, respectively. The mean recovery of the analyte was 99%. Derivatized samples demonstrated good short-term, long-term, post-preparative and freeze–thaw stability. This is the first reported LC–MS/MS method for analysis of captopril in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of the analyte. The validated LC–MS/MS method has been applied to a pharmacokinetic study of 50 mg captopril tablets on healthy volunteers.