Determination of dehydrodiisoeugenol in rat tissues using HPLC method

Dehydrodiisoeugenol (DDIE) is a bioactive neolignan from the seeds of Myristica fragrans Houtt., which exhibits good anti-inflammatory activity. A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of DDIE in rat tissues after intravenous administration. The tissue samples were processed by liquid-liquid extraction. The analyses were successfully carried out on a Diamonsiltrade mark ODS C18 column (250 x 4.6 mm i.d., 5 microm) equipped with a C18 guard column (8 x 4.6 mm i.d., 5 microm). The mobile phase was the system of methanol-water (4:1, v/v). The UV detection was set at 270 nm. The calibration curves were linear from 0.4 to 200.0 microg/g with the correlation coefficients (r(2)) greater than 0.998. The intra- and inter-day precisions in quality control samples were less than 10% and the accuracies were in the range 85.4-110.3%. The average recoveries from all the tissues were between 84.4 and 106.0%. This assay method has been successfully used to study the tissues distribution of DDIE in rats after intravenous administration. The result suggests that DDIE is distributed to rat tissues rapidly with possibly greater initial concentrations in liver and brain than in other tissues.     

HPLC method for determination of cefuroxime in plasma

Summary This paper describes an HPLC method for the determination of cefuroxime in human plasma. The method uses solid phase extraction (SPE) and has acceptable sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.1 μg mL⁻¹. Calibration curves were linear within 0.1–20 μg mL⁻¹, with mean correlation coefficient of 0.9982. Mean inter day precision and accuracy were 7.8% and 6.4%, respectively. The method was applied to determine cefuroxime levels in patients receiving cefuroxime, 3 time per day.     

LC-MS/MS method for the determination of cynandione A in rat plasma and tissues

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the quantitative determination of cynandione A in rat plasma and tissues. The plasma samples were pretreated by liquid-liquid extraction with ethyl acetate after the internal standard (honokiol) had been spiked. The tissue samples were homogenized with physiological saline and treated further like the plasma samples. The separation was performed using a Zorbax SB-C(18) column (3.5 microm, 2.1 x 100 mm) and a C18 guard column (5 microm, 4.0 x 2.0 mm) with an isocratic mobile phase consisting of methanol-0.1% formic acid (78:22, v/v) at a flow rate of 0.2 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode using the electrospray ionization technique in negative mode. The nominal retention times for cynandione A and honokiol were 1.41 and 2.63 min, respectively. The method was validated within the concentration range 0.2-1000 ng/mL in plasma and homogenized tissue for cynandione A, and the calibration curves were linear with correlation coefficients >0.992. The lower limit of quantification of cynandione A was 0.2 ng/mL. The intra-day and inter-day precision and accuracy of the assay in plasma were less than 14.4%, while the intra-day and inter-day precision and accuracy of the assay in tissue homogenate were less than 14.2%. This method proved to be suitable for study of pharmacokinetics and tissue distribution of cynandione A in rat.     

A simple HPLC method for simultaneous determination of lopinavir,ritonavir and efavirenz

We developed a simple HPLC method for the simultaneous determination of lopinavir (LPV), ritonavir (RTV) and efavirenz (EFV) to evaluate the efficiency of co-administration of LPV/RTV and EFV in Japanese patients enrolled in a clinical study. The monitoring of LPV plasma concentration is important because co-administration of LPV/RTV with EFV sometimes decreases plasma concentrations of LPV caused by EFV activation of cytochrome P-450 3A. A solution of acetonitrile, methanol and tetramethylammonium perchlorate (TMAP) in dilute aqueous trifluoroacetic acid (TFA) has been used as the mobile phase in a HPLC method to elute LPV and RTV. We found that a solvent ratio of 45 : 5 : 50 (v/v/v) of acetonitrile/methanol/0.02 M TMAP in 0.2% TFA optimized separation of LPV, RTV and EFV. A column temperature of 30 degrees C was necessary for the reproducibility of the analyses. Standard curves were linear in the range 0.060 to 24.06 micro g/ml for LPV, 0.010 to 4.16 micro g/ml for RTV, and 0.047 to 37.44 micro g/ml for EFV. Coefficients of variation (CVs) of LPV, RTV and EFV in intraday and interday assays ranged from 1.5 to 4.0%, 2.5 to 16.8% and 1.0 to 7.7%, respectively. Accuracies ranged from 100 to 110%, 101 to 116% and 99 to 106% for LPV, RTV and EFV, respectively. The extraction recoveries were 77-87, 77-83 and 81-91% for LPV, RTV and EFV, respectively.     

A simple isocratic HPLC method for the simultaneous determination of bioactive components of Scutellariae Radix extract

Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi, has been widely used in Asian countries for the treatment of dermatitis, diarrhoea, inflammatory disease and hepatic disease. A simple, sensitive and precise reversed-phase liquid chromatographic method with isocratic elution was developed to simultaneously determine four bioactive compounds in Scutellariae Radix: baicalein, baicalin, wogonin and wogonoside. Chromatographic analysis was performed on a YMC Pack Pro C(8) column (150?×?4.6?mm(2), 3?μm), with a mobile phase of 0.1% formic acid?:?acetonitrile (70?:?30, v/v) at a flow rate of 1.0?mL?min(-1), and UV detection at 280?nm. Linear behaviour was observed over the investigated concentration range (0.25-10?μg?mL(-1)) for all analytes, with a correlation coefficient of >0.997. The intra- and inter-day precisions were <8.07%, and accuracies were 92.3-102.9%. This method was successfully applied for the analysis of marker compounds for the quality control of Scutellariae Radix extract.     

Introduction of the HPLC method for the determination of quinolone residues in various muscle tissues

For use in veterinary sanitary control of foodstuffs and raw materials of animal origin in Slovenia, we developed a routine and confirmation analytical method for determining the residues of enrofloxacin, ciprofloxacin and flumequine in the muscle tissue of cattle, pigs and poultry. For the muscle tissue of freshwater fish, the determination of the flumequine residues was introduced. The results obtained through simultaneous determination of the residues of enrofloxacin, ciprofloxacin and flumequine showed that the values for the examined antibiotics were up to 600 times lower than the prescribed maximum residue levels (MRL). Another advantage of this method is that it covers a wide range of different fluoroquinolones.     

A simple method for simultaneous determination of basic dyes encountered in food preparations by reversed-phase HPLC

The present method utilizes a simple pretreatment step, cleanup on polyamide SPE cartridges, and HPLC resolution on reversed-phase C18 for the detection of the three basic nonpermitted dyes encountered in food matrixes. Polyamide cartridges were chosen because both acidic and basic dyes can be cleaned up due to their amphoteric nature. Analysis was performed on a reversed-phase C18 micro-Bondapak column using the isocratic mixture of acetonitrile-sodium acetate with a flow rate of 1.5 mL/min and a programmable lambda(max) specific visible detection to monitor colors, achieving higher sensitivity and expanded scope to test multicolor blends. All the colors showed linearity with the regression coefficient, from 0.9983 to 0.9995. The LOD and LOQ ranged between 0.107 and 0.754 mg/L and 0.371 and 2.27 mg/L or mg/kg, respectively. The intraday and interday precision gave good RSDs, and percentage recoveries in different food matrixes ranged from 75 to 96.5%. The study demonstrates that the use of a combination of a simple SPE cleanup and HPLC resolution with UV-Vis end point detection was successful in screening the presence of these three basic nonpermitted dyes individually or in blend, in a variety of food matrixes.     

A method for the determination of d-kynurenine in biological tissues

d-Kynurenine (d-KYN), a metabolite of d-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of d-KYN in biological tissues, we developed a novel assay based on the conversion of d-KYN to KYNA by purified d-amino acid oxidase (d-AAO). Samples were incubated with d-AAO under optimal conditions for measuring d-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for d-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other d-amino acids, including d-serine and d-aspartate, were present in the incubation mixture at 50-fold higher concentrations than d-KYN. Using this new method, d-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with d-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for d-KYN measurement will be instrumental for evaluating whether d-KYN should be considered for a role in physiology and pathology.     

A simple spectrophotometric method for the determination of minoxidil

A simple and sensitive method for the determination of minoxidil using 3-methyl-2-benzothiazolinone hydrazone (MBTH) and Fe(III) as the coloring agent is described. The method is sensitive to permit the determination of 0.5–4.5 μg ml⁻¹ of minoxidil at 500 nm. The accuracy and precision of the method were checked by high-performance liquid chromatography.     

A method for the determination of histamine in wine by HPLC with precolumn derivatization with phenylisothiocyanate

Summary A method for determining histamine in wine by precolumn derivatization with PITC (phenylisothiocyanate) with reversed-phase HPLC and UV detection is reported. Histamine can be determined together with the 24 amino acids within 40 min, or separately in a shorter time (less than 4 min) if a prior solid phase extraction clean-up is used.     

A validated solid-liquid extraction method for the HPLC determination of polyphenols in apple tissues Comparison with pressurised liquid extraction

A solid-liquid extraction procedure followed by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector (DAD) for the determination of polyphenols in freeze-dried apple peel and pulp is reported. The extraction step consists in sonicating 0.5 g of freeze-dried apple tissue with 30 mL of methanol-water-acetic acid (30:69:1, v/v/v) containing 2 g of ascorbic acid/L, for 10 min in an ultrasonic bath. The whole method was validated, concluding that it is a robust method that presents high extraction efficiencies (peel: >91%, pulp: >95%) and appropriate precisions (within day: R.S.D. (n = 5) <5%, and between days: R.S.D. (n = 5) <7%) at the different concentration levels of polyphenols that can be found in apple samples. The method was compared with one previously published, consisting in a pressurized liquid extraction (PLE) followed by RP-HPLC-DAD determination. The advantages and disadvantages of both methods are discussed.     

A general method for the quantitative determination of catecholamines in body fluids using off-line sample pretreatment and HPLC-ED analysis

Summary Reversed phase HPLC with electrochemical detection (HPLC-ED) was used for quantitative determination of adrenaline, noradrenaline, and dopamine in several complex biological matrices, including plasma, uremic plasma, and urine. Three different methods of sample preparation for use in this clinical chemistry were tested. These were adsorption of catecholamines on alumina, organic solvent extraction after complex formation with diphenylborate, and adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute. The selectivity and precision of the three methods were evaluated. The organic solvent extraction proved to be more precise and selective than adsorption on alumina (adrenaline: cv=3.80% vs. 7.58%; noradrenaline: cv=1.70% vs. 4.26%); it also proved suitable for use in the routine quantitative determination of catecholamines in plasma from patients with normal renal function (creatinine <1.2 mg/dl). However when working with uremic plasma or urine, a more selective sample preparation was required. In this case the adsorption of catecholamines on a cation exchange gel followed by organic solvent extraction of the elute was sufficiently selective and precise and thus allowed a reliable quantitative determination of adrenaline and noradrenaline from rather complex biological matrices (adrenaline: cv=6.2%; noradrenaline: cv=2.8%). Use of this specific method showed that basal plasma catecholamine levels in dialysis patients are comparable to those found in patients with normal renal function (adrenaline: 47.7±22.2 pg/ml; noradrenaline: 310.3±121.4 pg/ml).     

A simple colorimetric method for the determination of ammonia in seawater

A method is proposed for the determination of trace amounts of ammonia in seawater. After calcium and magnesium have been chelated with CDTA, the blue colour obtained with hypochlorite and thymol-acetone is measured at 630 nm. The sensitivity is 1.3 ng NH₄⁺-N/cm².     

A simple HPLC method for quantitation of quercetin in herbal extracts

A reversed-phase HPLC method with UV detection was developed for the determination of quercetin. The method produced linear response over a wide concentration range, with an average accuracy of 95% and average intra- and interday variation of 0.75 and 0.3, respectively. The exactness of the method was proven by determining the recovery rates from 50 to 150% of standard concentration, which were found within the acceptable range of 95 to 105%. The method was used for quantitation of quercetin in the extracts of Psidium guajava, Vitis vinifera, and extracts rich in quercetin and other flavonols in the flavonoid family.