HPLC-MS/MS method for determination of betamethasone in human plasma with application to a dichorionic twin pregnancy pharmacokinetic and placental transfers studies

Abstract Betamethasone (BET) is a synthetic glucocorticoid recommended for pregnant women at imminent risk of preterm birth before 34 weeks to reduce neonatal complications. There are different techniques to describe BET plasma quantification. However, none quantified the plasmatic concentration of BET in dichorionic (DC) twin pregnancies using LC-MS. Our objectives were to develop and validate a method for quantifying BET by LC-MS for pharmacokinetic (PK) and placental transfer studies in DC twin pregnancies. Blood samples were collected after intramuscular administration of a single BET dose containing 6 mg disodium phosphate + 6 mg acetate. BET was determined in plasma by liquid-liquid extraction. The method showed linearity in the range of 2-250 ng/mL, as well as precision and accuracy with a coefficient of variation and relative standard errors ≤ 15%. Additionally, the method presented selectivity and did not present matrix or carry-over effect. Stability tests also presented coefficient of variation and relative standard errors ≤ 15%. This is the first study which describe maternal and fetal plasma concentrations of BET in a DC twin pregnancy. The BET PK parameters were AUC0-∞, CL/F, Vd/F, Cmax, Tmax of 292.20 h*ng/mL, 39.08 L/h, 278.72 L, 25.55 ng/mL and 0.58 h, respectively. The placental transfer ratios of umbilical vein/maternal vein and intervillous space/maternal vein were 0.14 and 0.19 and 0.40 and 0.27 for both twins, respectively. However, a clinical study with more subjects is imperative to confirm this higher concentration of BET in the intervillous space

Ion Abundance Ratios of Qualitative Analysis by GC-MS of 4 Common Drugs （Poisons / 法医学杂志

Objective To investigate the maximum allowable deviation of ion abundance ratios of characteristic fragment ions in common drugs （poisons） in blood by gas chromatography-mass spectrometry （GC-MS） method. Methods Four common drugs （poisons） （dichlorvos, phorate, diazepam and estazolam） were detected by GC-MS full scan mode after liquid-liquid extraction in two laboratories and under three chromatographic conditions. The deviations of ion abundance ratios of the four common drugs （poisons） in marked blood samples with concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 μg/mL were analyzed. At the same time, the false negative rates of ion abundance ratios were analyzed when the mass concentration was limit of detection （LOD）, 2LOD, limit of quantitation （LOQ） and 2LOQ, and the false positive rates of ion abundance ratios were analyzed with blank blood samples. Results Under the two laboratories, four common drugs （poisons） and three kinds of chromatography conditions, the differences in deviations of the ion abundance ratios of marked blood samples were not statistically significant （P>0.05）. More than 95% of the absolute deviations of the ion abundance ratios of the marked blood samples were within the range of ±10%, and more than 95% of the relative deviations were within the range of ±25%. In cases of low concentration （concentration less than 2LOQ） or low signal to noise ratio （3-15）, the false negative rate was less than 5% and the false positive rate was 0% when the relative deviation was greater than 50%. Conclusion The absolute deviations of ion abundance ratios of four common drugs （poisons） in marked blood samples are advised to have a determination range within ±10%, and the determination range of relative deviations within ±25%.

Removing Lipemia in Serum/Plasma Samples: A Multicenter Study

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods—LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

Determination of donepezil in human plasma using ultra performance liquid chromatography-tandem mass spectrometry

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of donepezil in human plasma. Donepezil and donepezil-D4 were extracted from human plasma by liquid-liquid extraction using a mixture of hexane and ethyl acetate (70:30 v/v). The extracted samples were analyzed using a Thermo Hypersil Gold C18 column with 5% acetic acid in 20 mM ammonium acetate buffer (pH 3.3) and 100% acetonitrile as a mobile phase with the 60:40 (v:v) isocratic method, at a flow rate of 0.3 mL/min. The injection volume was 3 µL, and the total run time was 3 min. Inter- and intra-batch accuracies ranged from 98.0% to 110.0%, and the precision was below 8%. The developed method was successfully applied to the quantification of donepezil in human plasma. The mean (standard deviation) maximum concentration and the median (range) time to maximum concentration were 8.6 (2.0) ng/mL and 2.0 h (1.0~5.0 h), respectively, in healthy Koreans after oral administration of 5 mg donepezil.

Safety, Efficacy, and Physicochemical Characterization of Tinospora crispa Ointment: A Community-Based Formulation against Pediculus humanus capitis

The high prevalence of pediculosis capitis, commonly known as head lice (Pediculus humanus capitis) infestation, has led to the preparation of a community-based pediculicidal ointment, which is made of common household items and the extract of Tinospora crispa stem. The present study aimed to evaluate the safety, efficacy, and physicochemical characteristics of the T. crispa pediculicidal ointment. The physicochemical properties of the ointment were characterized, and safety was determined using acute dermal irritation test (OECD 404), while the efficacy was assessed using an in vitro pediculicidal assay. Furthermore, the chemical compounds present in T. crispa were identified using liquid-liquid extraction followed by ultra-performance liquid chromatography quadruple time-of-flight mass spectrometric (UPLC-qTOF/MS) analysis. The community-based ointment formulation was light yellow in color, homogeneous, smooth, with distinct aromatic odor and pH of 6.92±0.09. It has spreadability value of 15.04±0.98 g·cm/sec and has thixotropic behavior. It was also found to be non-irritant, with a primary irritation index value of 0.15. Moreover, it was comparable to the pediculicidal activity of the positive control Kwell®, a commercially available 1% permethrin shampoo (P>0.05), and was significantly different to the activity of the negative control ointment, a mixture of palm oil and candle wax (P<0.05). These findings suggested that the community-based T. crispa pediculicidal ointment is safe and effective, having acceptable physicochemical characteristics. Its activity can be attributed to the presence of compounds moupinamide and physalin I.

Development of a simple and sensitive HPLC-MS/MS method for determination of diazepam in human plasma and its application to a bioequivalence study

We developed a simple, sensitive, and effective ultra-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method with an electrospray ionization (ESI) interface in multiple reaction monitoring (MRM) and positive ion modes to determine diazepam concentrations in human plasma using voriconazole as an internal standard (IS). Diazepam and IS were detected at transition 285.2→193.1 and 350.2→127.1, respectively. After liquid-liquid extraction (LLE) using 1.2 ml of ethyl acetate:n-hexane (80:20, v/v), diazepam and IS were eluted on a Phenomenex Cadenza CD-C18 column (150 × 3.0 mm, 3 µm) with an isocratic mobile phase (10 mM ammonium acetate in water:methanol [5:95, v/v]) at a flow rate of 0.4 mL/min. The peak retention time was 2.32 min for diazepam and 2.01 min for IS, respectively. The lower limit of quantitation (LLOQ) was 0.5 ng/mL (S/N > 10) using 50 µL of plasma, and no interferences were observed in chromatograms. Our analytical method was fully validated and successfully applied to a bioequivalence study of two formulations of diazepam in healthy Korean volunteers.

Development and validation of analytical method for the determination of radotinib in human plasma using liquid chromatography-tandem mass spectrometry

This study describes the development of an analytical method to determine radotinib levels in human plasma using high performance liquid chromatography (HPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) for pharmacokinetic application. Plasma samples were sequentially processed by liquid-liquid extraction using methyl tert-butyl ether, evaporation, and reconstitution. Analytes were separated and analyzed using HPLC-MS/MS in selected reaction monitoring mode, monitoring the specific transitions of m/z 531 to 290 for radotinib and m/z 409 to 238 for amlodipine (internal standard). The HPLC-MS/MS analytical method was validated with respect to selectivity, linearity, sensitivity, accuracy, precision, recovery, and stability. Calibration curves were linear over a concentration range 5–3,000 ng/mL with correlation coefficients (r) > 0.998. The lower limit of quantification for radotinib in plasma was 5 ng/mL. The accuracy and precision of the analytical method were acceptable within 15% at all quality control levels. This method was suitable to determine radotinib levels in human plasma because of its simplicity, selectivity, precision, and accuracy.

Effective method for the detection of piroxicam in human plasma using HPLC

Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5'-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC) after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo - USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg) and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5'-hydroxypiroxicam was only detected in 2 out of 10 volunteers.

Antioxidant activities of crude phlorotannins from Sargassum hemiphyllum / 华中科技大学学报（医学）（英德文版）

Brown algae are well known as a source of biologically active compounds, especially those having antioxidant activities, such as phlorotannins. In this study we examined the antioxidant activities of crude phlorotannins extracts (CPEs) obtained from Sargassum hemiphyllum (SH) and fractionated according to the molecular weights. When CPEs were administrated at a dose of 30 mg/kg to Kunming mice pre-treated with carbon tetrachloride (CCl4), the levels of oxidative stress indicators in the liver, kidney and brain were significantly reduced in vivo. All the components of various molecular weight fractions of CPEs exhibited greater scavenging capacities in clearing hydroxyl free radical and superoxide anion than the positive controls gallic acid, vitamin C and vitamin E. Particularly, the components greater than 30 kD obtained from ethyl acetate phase showed the highest antioxidant capacities. These results indicated that SH is a potential source for extracting phlorotannins, the algal antioxidant compounds.

Development of a validated liquid chromatography-tandem mass spectrometry assay for the quantification of simvastatin acid, the active metabolite of simvastatin, in human plasma

Simvastatin is a lipid-lowering drug that is metabolized to its active metabolite simvastatin acid (SA). We developed and validated a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to quantitate SA in human plasma using a liquid-liquid extraction method with methanol. The protonated analytes generated in negative ion mode were monitored by multiple reaction monitoring. Using 500-mL plasma aliquots, SA was quantified in the range of 0.1-100 ng/mL. Calibration was performed by internal standardization with lovastatin acid, and regression curves were generated using a weighting factor of 1/χ2. The linearity, precision, and accuracy of this assay for each compound were validated using quality control samples consisting of mixtures of SA (0.1, 0.5, 5, and 50 ng/mL) and plasma. The intra-batch accuracy was 95.3-107.8%, precision was -2.2% to -3.7%, and linearity (r2) was over 0.998 in the standard calibration range. The chromatographic running time was 3.0 min. This method sensitively and reliably measured SA concentrations in human plasma and was successfully used in clinical pharmacokinetic studies of simvastatin in healthy Korean adult male volunteers.

Simultaneous screening for 45 poisonous alkaloids in blood by LC-MS/MS / 法医学杂志

OBJECTIVE@#To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for 45 poisonous alkaloids in blood.@*METHODS@#Identification was based on the compound's retention time and two precursor-to-production transitions. The method involved a liquid-liquid extraction (LLE) followed by LC-MS/MS with multiple-reaction monitoring (MRM). When 1 mL of blood was extracted with diethyl ether at pH = 9.2 with SKF525A as the internal standard, the target compounds were analyzed with LC-MS/MS in the positive ionization mode.@*RESULTS@#The target alkaloids had good linearity (r>0.995 1), both the intra-day precision and inter-day precision being less than 14.77%. The limits of detection ranged from 0.05 to 25 ng/mL in blood.@*CONCLUSION@#The method is selective and sensitive in detecting poisonous alkaloids with a total running time of 12 minutes; therefore it was successfully applied to some actual cases of suspected alkaloids poisoning.

Quality control in liquid-liquid extraction of Reduning injection using near-infrared spectroscopy technology / 中国中药杂志

Quantitative models were established to analyze the content of chlorogenic acid and soluble solid content in the liquid-liquid extraction of Reduning injection by near-infrared (NIR) spectroscopy. Seven batches of extraction solution from the liquid-liquid extraction of Lonicerae Japonicae Flos and Artemisiae Annuae Herba were collected and NIR off-line spectra were acquired. The content of chlorogenic acid and soluble solid content were determined by the reference methods. The partial least square (PLS) and artificial neural networks (ANN) were used to build models to predict the content of chlorogenic acid and soluble solid content in the unknown samples. For PLS models, the R2 of calibration set were 0.9872, 0.9812, RMSEC were 0.1533, 0.7943, the R2 of prediction set were 0.9837, 0.9733, RMSEP were 0.2464, 1.2594, RSEP were 3.25%, 3.31%, for chlorogenic acid and soluble solid content, respectively. For ANN models, the R2 of calibration set were 0.9903, 0.9882, RMSEC were 0.0974, 0.4543, the R2 of prediction set were 0.9868, 0.9699, RMSEP were 0.1920, 0.9427, RSEP were 2.61%, 2.75%, for chlorogenic acid and soluble solid content, respectively. Both the RSEP values of chlorogenic acid and soluble solid content were lower than 6%, which can satisfy the quality control standard in the traditional Chinese medicine production process. The RSEP values of ANN models were lower than PLS models, which indicated the ANN models had better predictive performance for chlorogenic acid and soluble solid content. The established method can rapidly measure the content of chlorogenic acid and soluble solid content. The method is simple, accurate anc reliable, thus can be used for quality control of the liquid-liquid extraction of Reduning injection.

Quantification of sitagliptin in human plasma and urine by LC-MS/MS method and its application / 药学学报

A rapid and sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for quantification of sitagliptin in human plasma and urine had been developed. This method was applied to the pharmacokinetics study of sitagliptin tablet after single- and multiple-dosing in Chinese population. Plasma samples were prepared by a liquid-liquid extracted method, and urine samples were diluted. Compounds were analyzed by multiple reaction monitoring (MRM) mode with a electrospray ionization (ESI) interface. Mobile phase consisted of methanol and water (85 : 15, v/v). The linear concentration range of calibration curve was 0.5-1 000 ng.mL-1. and 0.2-100 µg.mL , intra-run/between-run accuracy was 98.98%-103.69% and 97.63%-102.29%, intra-run/between-run precision was <5.51% and 4.26% for plasma and urine sample, respectively. The stability of sitagliptin stock solution was tested for 55 days at -30 °C. Sitagliptin was stable when stored under the following conditions: 24 hours in the autosampler after sample preparation; 24 hours at room temperature, after 3 freeze and thaw cycles (from -30 °C to room temperature), 40 days at -30 °C for plasma and urine samples. The absolute recovery in plasma was 71.1%, and no matrix effect was founded. This method was proved simple, specific, sensitive, rapid and suitable for pharmacokinetics study of sitagliptin in human being.

Acute oral toxicity and anti-inflammatory activity of hydroalcoholic extract from Lampaya medicinalis Phil in rats

BACKGROUND: Algesia and inflammation are related with several pathological conditions. It is known that many drugs available for the treatment of these problems cause unwanted side effects. This study was aimed at evaluating acute toxicity and anti-inflammatory activity of Lampaya medicinalis Phil. (Verbenaceae) widely used in the folk medicine of Northern Chile against rheumatism, arthritis and body joints pain. RESULTS: Oral administration of hydroalcoholic extract (HAE) at the highest dose of 3000 mg/ Kg body weight resulted in no mortalities or evidence of significant behavioral changes. Histological examination revealed normal architecture and no significant adverse effects were observed on the liver, kidney, heart, lung or ovaries and testicles. The results suggest that the oral administration of hydroalcoholic extract (HAE) from Lampaya medicinalis did not produce any toxic effect in rats. Hydroalcoholic extract (HAE) significantly inhibited the carrageenan-induced rat paw edema in dose - response relationship, at test doses of 37.5, 75, 150 and 300 mg/Kg body weight. Maximum inhibition (61.98 ± 2.69%) was noted at 300 mg/Kg after 2 h of drug treatment carrageenan induced paw edema, whereas indomethacin produced 47.90 ± 1.16% of inhibition. The inhibitory values of edema at 3 h postcarrageenan were 31.04±0.75%, 40.51 ± 2.36%, 48.97 ± 1.14% and 56.87 ± 0.41% for 37.5, 75, 150, and 300 mg/kg of extract respectively. Indomethacin (10 mg/Kg) gave a percentage inhibition of 49.44 ± 1.44. HAE (300 and 150 mg/kg) induced an anti-inflammatory effect greater than (or comparable) with the effect of indomethacin from 2nd to 4th hours of the experiment. CONCLUSIONS: Our results reveal for first time that compounds contained in the hydroalcoholic extract ofLampaya medicinalis Phil exert anti-inflammatory effect and the oral administration is safe and non toxic up to dose level 3000 mg/kg body weight. The anti-inflammatory activity may be associated with the presence of flavonoids. These findings also justify the traditional use of the plant for treating pain.

Residue decline dynamics and safety utilization of carbendazim in cultivation of Anoectochilus roxburghii / 中国中药杂志

The paper aimed to study the residue decline dynamic and standards for safety utilization of carbendazim in roots, stems, leaves of Anoectochilus roxburghii and in growth media. Samples extracted with methanol were purified by liquid-liquid extraction and analysed by HPLC. The results showed that average rate of recovery was 82.9% - 95.7% and RSD were 2.0% - 6.3% with add of carbendazim in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of carbendazim were treated, varying from recommended dosage (1.0 kg x hm(-2)) to 1.5 times recommended dosage (1.5 kg x hm(-2)). Results of two years test showed that the half-life period of carbendazim were 7.01 - 8.51 d in the growth media of A. roxburghii, 3.58 - 4.27 d in stems and 3.50 - 3.91 d in leaves, 4.93 - 5.71 d in roots. Providing max recommended residue of carbendazim in the cultivation of A. roxburghii is 0.5 mg x kg(-1), sprayed 4 times a year with the dosage of 1.0 kg x hm(-2), 28 days is proposed for the safety interval of the last pesticide application's and harvest's date.

Research progress of pretreatment of biological samples / 中国中药杂志

Suitable pretreatment of biological samples can truly reflect the role of law of the measured components played in the body and will provide experimental evidence for the studies on metabolic process, material basis of efficacy, mechanism of action, pharmacology, toxicology and the others. Biological samples include blood, urine, hair, tears, etc. There are also many samples processing methods, such as the direct protein precipitation, liquid-liquid extraction and solid phase extraction and so on. These methods could be used alone or combined.

Development of a LC-MS/MS for Quantification of Venlafaxine in Human Plasma and Application to Bioequivalence Study in healthy Korean Subjects

A simple, rapid and selective liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is developed and validated for quantification of venlafaxine in human plasma with simple liquid-liquid extraction step consisted of extraction with ether and dichloromethane for 10 min and mixing with 1 M sodium acetate in human plasma using fluoxetine as an internal standard (IS). The analyte are separated using an isocratic mobile phase consisted of acetonitrile and 5 mM ammonium formate (4/3, v/v) on a isocratic YMC hydrosphere C18 (2.0x50.0 mm, 3.0 microm) column and analyzed by MS/MS in the multiple reaction monitoring (MRM) mode using the transitions of respective [M+H](+) ions, m/z 278.2-->260.3 and m/z 310.1-->148.1 for quantification of venlafaxine and IS, respectively. The standard calibration curves showed good linearity within the range of 1.0-200.0 ng/mL (r2=0.9986, 1/chi2 weighting). The lower limit of quantification (LLOQ) was 1.0 ng/mL. The retention times of venlafaxine and IS were 0.6 min and 0.7 min that means the potential for the high-throughput potential of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. Acceptable precision and accuracy were obtained for the concentrations over the standard curve range. The validated method was successfully applied to bioequivalence study after 75-mg of venlafaxine sustained-release (SR) capsule in 24 healthy Korean subjects.

Produção e extração de colorantes naturais de Penicillium purpurogenum DPUA 1275 / Production and extraction of natural colorants from Penicillium purpurogenum DPUA 1275

Há interesse mundial no desenvolvimento de pesquisas envolvendo produção e extração de colorantes naturais, devido a sérios problemas de segurança industrial associados ao uso de colorantes sintéticos. Este trabalho objetivou produzir colorantes naturais de Penicillium purpurogenum DPUA 1275 por cultivo submerso (em frascos agitados e em biorreator) e estudar a extração dos colorantes vermelhos. Para a produção, os estudos iniciais mostraram que 5 discos de micélio, sacarose e extrato de levedura como fontes de carbono e nitrogênio, respectivamente, e 336 horas de cultivo eram condições adequadas para a produção dos colorantes. Visando à otimização da produção, realizaram-se planejamentos fatoriais, com as variáveis independentes: tempo de cultivo; velocidade de agitação; pH; temperatura; concentração de sacarose e de extrato de levedura. As variáveis-respostas foram produção de colorantes amarelos, laranjas e vermelhos. Dos resultados obtidos, as variáveis mais significativas ao processo foram concentrações de extrato de levedura e de sacarose. A produção dos colorantes vermelhos foi otimizada, alcançando a produção de 2,97 UA490nm, nas condições 48,90 e 11,80 g/L de sacarose e extrato de levedura, respectivamente, 30°C, pH 4,5 150 rpm e 336 horas de cultivo. Nos experimentos em biorreator, o melhor resultado foi obtido na frequência de agitação de 500 rpm e na mudança do pH do meio para 8,0, após 96 horas de bioprocesso. Ademais, avaliou-se a estabilidade dos colorantes vermelhos presentes no meio fermentado em diferentes condições (pH, temperatura, sais, polímeros e tensoativos). Referente a pH e temperatura, os colorantes vermelhos mostraram-se mais estáveis nas condições alcalinas e a 70 °C. Tanto os sais (NaCl e Na2SO4) quanto os polímeros (PEG 1.000, 6.000 e 10.000 g/mol e NaPA 8.000 g/mol a 5 e 15%) e os tensoativos (Tween 20, CTAB e SDS) não causaram perda da cor nas condições avaliadas. Estudos de solubilidade e de coeficiente de partição octanol-água mostraram que os colorantes vermelhos apresentam solubilidade superior em solventes polares e característica mais hidrofílica. Nos estudos de extração, as técnicas avaliadas foram Sistemas Poliméricos de Duas Fases Aquosas (SPDFA) formados pelo sistema PEG/NaPA e Colloidal Gas Aphrons (CGA). Pela primeira técnica, os colorantes vermelhos migraram preferencialmente para a fase PEG. Os polímeros PEG 6.000 g/mol, na presença de NaCl 0,1 e 0,5 M, e PEG 10.000 g/mol, com Na2SO4 0,5M, se destacaram dentre as condições analisadas com coeficiente de partição (K) próximo a 13, em ambos os casos, e seletividade de proteínas (SeP) próximas a 3. Para a técnica de CGA, o CTAB proporcionou os melhores resultados, seguido do Tween 20. Porém, o valor de K foi inferior ao obtido com SPDFA, com um máximo de 5 (CTAB 2 mM/pH 9,0). Os resultados obtidos demonstram um novo produtor de colorantes naturais, as quais têm potencial de aplicação em diversos segmentos industriais. Ademais, os resultados obtidos mostraram a eficiência das técnicas utilizadas para extração dos colorantes vermelhos, com destaque para SPDFA, que apresentou maiores valores de K

There is worldwide interest in developing research projects involving the production and extraction of natural colorants due to serious safety problems associated with industrial use of synthetic ones. The aim of this work was to investigate the production of natural colorants from Penicillium purpurogenum DPUA 1275 by submerged culture (rotatory shaker and bioreactor) besides studying the red colorants extraction. To the production step, initial studies showed that 5 agar mycelial discs, sucrose and yeast extract as carbon and nitrogen sources, respectively, and 336 hours of bioprocess promoted the best results. To optimize the colorants production a serie of factorial designs were performed. The independent variables studied were: fermentation time, agitation speed, pH, temperature, sucrose and yeast extract concentration under the responses production of yellow, orange and red colorants. From these results, the most significant variables for the process were sucrose and yeast extract concentration. The red colorants production was optimized achieving 2.97 UA490nm, in the following conditions: 48.90 and 11.80 g/L of sucrose and yeast extract, respectively, 30 °C, 4.5 pH, 150 rev min-1 and 336 hours of culture. In the experiments performed in bioreactor, the condition that promoted the best results was 500 rpm and pH adjusted for 8.0 after 96 hours of bioprocess. Furthermore, we evaluated the red colorants stability at different conditions (pH, temperature, salts, polymers and surfactants). Concerning to pH and temperature, the red colorants were more stable under basic conditions and 70 °C; not only the salts (NaCl and Na2SO4) but also the polymers (PEG 1000, 6000 and 10000 g/mol and NaPA 8000 g/mol) and the surfactants (Tween 20, CTAB and SDS) not promoted loss of color upon the conditions evaluated. Studies of red colorants solubility and octanol water coefficient showed that these compounds exhibit a higher solubility in polar solvents and present hydrophilic characteristics. Subsequently, the extraction of red colorant was evaluated through two extraction methods: Polymeric Systems Aqueous Two Phase (ATPS) composed by PEG and NaPA and Colloidal Gas Aphrons (CGA). For the first technique, the red colorant preferentially migrated to the PEG phase. The best results were obtained with PEG 6000 g/mol in the presence of 0.1 to 0.5 M NaCl and with PEG 10000 g/mol with 0.5 M Na2SO4. To both cases the partition coefficient (K) was close to 13 and the Selectivity in terms of proteins (SeP) was close to 3. For the CGA technique, CTAB gave the best results followed by Tween 20. However, the K values were lower than the ones obtained with ATPS with a maximum of 5 in the following condition: CTAB 2 mM/pH 9.0. For the SeP, the values obtained for both techniques were close. The results above show a new producer of natural colorants which have potential application in various industries. Moreover, the results show the efficiency of the techniques used to extract the red colorants, especially to ATPS that presented higher K values

Development and validation of high performance liquid chromatographic method for analysis of clozapine

In this study a rapid, simple and sensitive assay to quantify clozapine in human plasma by using reverse phase high performance liquid chromatographic method has been developed. Clozapine was extracted from human plasma using a mixture of chloroform: n-hexane 50:50 employing liquid- liquid extraction method. The calibration curve was found to be linear in the concentration range of 25-800 ng/ml. The inter day and intra day assay accuracy and precision fulfilled the criteria specified by USFDA, Guidance for industry: bioanalytical method validation. Clozapine was found to be stable in human plasma after 6 h incubation at room temperature, 50 days storage at -27[degree sign] C and freeze thaw cycles, as well as after reconstitution with mobile phase after 24 h of storage in refrigerator. The validated method offers the advantage of using minimum injection volume [25micro l] and plasma sample volume [300micro l]. The extraction method is simple and single step with no back extraction step, thus, making this method applicable to determination of pharmacokinetic profiles and parameters

Butanol production from corn stover hydrolysate with in-situ liquid-liquid extraction / 生物工程学报

Butanol production from corn stover hydrolysates (CSH) with in-situ liquid-liquid extraction was studied to enhance the production and reduce the fermentation cost. Oleyl alcohol was selected as the suitable solvent and added at the initial fermentation time with the ratio of 1:1 (oleyl alcohol: fermentation broth, V/V). Under this condition, butanol and ABE from CSH with 32.1 g/L total sugars were 3.28 and 4.72 g/L, which were 958.1% and 742.9% higher than those of the controls, respectively. Butanol and ABE production from CSH of 49.7 g/L total sugars after detoxification by ion exchange resin D301 coupled with extraction fermentation were 10.34 g/L and 14.72 g/L with an ABE yield of 0.31 g/g (g ABE/g utilized sugar), which were equal to those of glucose and xylose mixture fermentation. The detoxification and extraction fermentation technology of cellulosic butanol production would provide a crucial technical support to the industrialized production of cellulosic butanol.