On-line coupling of dynamic microwave-assisted extraction with high-performance liquid chromatography for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees

A novel technique based on dynamic microwave-assisted extraction (DMAE) coupled on-line with high-performance liquid chromatography (HPLC) through a flow injection interface has been developed for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees. A TM(010) microwave resonance cavity built in the laboratory was applied to concentrating the microwave energy. An extraction vessel was placed in microwave irradiation zone. The extraction was performed in a recirculating system. When a number of extraction cycles were completed, the fractional extract (20muL) was driven to the analytical column by 65% aqueous methanol and was measured by diode array detector (DAD) at 225nm. The optimized extraction conditions are follows: extraction solvent 60% aqueous methanol; microwave forward power 80W; extraction time 6min; extraction solvent flow-rate 1.0mLmin(-1). The detection and quantification limits obtained are 0.5 and 1.7microgmL(-1) for andrographolide and 0.6 and 1.9microgmL(-1) for dehydroandrographolide, respectively. The within-day and between-day precision (RSD) are 2.1% and 3.7% for andrographolide and 1.7% and 4.1% for dehydroandrographolide, respectively. Mean recoveries for andrographolide and dehydroandrographolide are 97.7% and 98.7%, respectively. Compared with ultrasonic extraction used in the Chinese pharmacopoeia, the proposed method was demonstrated to obtain higher extraction yield in a shorter time. In addition, only small quantities of solvent (5mL) and sample (10mg) were required.     

Simultaneous determination of four bufadienolides in human liver by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous quantitation of four bufadienolides-cinobufotalin, bufalin, cinobufagin and resibufogenin-in human liver was investigated. The procedure involved solid phase extraction of human liver with an Oasis HLB cartridge coupled with reversed-phase HPLC and photodiode array detection. Recoveries obtained from spiked liver for the bufadienolides were better than 70%. The linearity was studied up to 1.2 mg/kg and the detection limits of the method were 0.4 ng for cinobufotalin and bufalin and 0.5 ng for cinobufagin and resibufogenin. The developed method was successfully applied to a lethal poisoning case.     

Simultaneous determination of four antiepileptic drugs in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin. After protein precipitation by acetonitrile, the supernatant was analysed on a C18 reversed-phase HPLC column. Antiepileptic drugs and oxazepam (internal standard) were detected by ultraviolet absorbance at 240 nm. Linearity was established for the whole concentration range for each compound. Quantitation limits of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin were 0.58, 3.5, 2.35, 0.66, 1.02 and 3.13 microg/mL, respectively, and mean recoveries added to serum were 105.15, 84.76, 94, 45, 96.52, 98.62 and 95.08%, respectively. This method has been used for the simultaneous determination of steady-state serum concentration of antiepileptic drugs in patients treated by one or more anticonvulsive treatment.     

Simultaneous determination of alpha-tocopherol and beta-carotene in olive oil by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed for the determination, in one run, of alpha-tocopherol and beta-carotene in virgin olive oil. The method involved a rapid saponification and a later extraction with a mixture of hexane-ethyl acetate. The chromatographic system consists of an ODS-2 column with a mobile phase of methanol-water-butanol and a diode-array detector. Linearity, precision, recovery and sensitivity were satisfactory. The main advantage of the proposed method is the speed and simultaneous determination of both compounds at the same time.     

Simultaneous determination of R- and S-prenylamine in plasma and urine by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of R- and S-prenylamine in human plasma and urine is described. It involves a two-step liquid-liquid extraction of prenylamine from biological material and preparation of diastereomeric urea derivatives with R-(-)-naphthylethyl isocyanate, a chiral fluorescence marker. Separation and quantitation of the diastereomeric prenylamine derivatives are carried out by a reversed-phase high-performance liquid chromatographic system with fluorimetric detection. The limit of determination is less than 2 ng of enantiomer per ml of urine and less than 1 ng of enantiomer per ml of plasma. A preliminary kinetic study on one healthy volunteer who had received a single oral dose of racemic prenylamine (100-mg film tablet) showed distinctly higher plasma and urine concentrations of the R-enantiomer.     

Simultaneous determination of 11 drugs belonging to four different groups in human urine samples by reversed-phase high-performance liquid chromatography method

A new, accurate, sensitive and fast reversed-phase high-performance liquid chromatography (RP-HPLC) as an analytical method for the quantitative determination of 11 drugs in human urine was worked out, optimized and validated. The objects of analysis were imipenem (IMP), paracetamol (PAR), dipyrone (DPR), vancomycin (VCM), amikacin (AMK), fluconazole (FZ), cefazolin (CFZ), prednisolone (PRE), dexamethasone (DEX), furosemide (FUR) and ketoprofen (KET) belonging to four different groups (antibiotics, analgesic, demulcent and diuretic). For HPLC analysis, diode array (DAD) and fluorescence (FL) detectors were used. The separation of analyzed compounds was conducted by means of a LiChroCART^® Purospher^® C₁₈e (125 mm × 3 mm, particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) pre-column with gradient elution. Analyzed drugs were determined within 20 min. The mobile phase was comprised of various proportions of methanol, acetonitrile and 0.05% trifluoroacetic acid in water. AMK was separated and determined from human urine using ortho-phthaldialdehyde-3-mercaptopropionic acid (OPA-3-MPA) as a fluorescent reagent by RP-HPLC-FL. The following retention times for drugs IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET in human urine were found: 4.01 min, 4.86 min, 6.71 min, 8.14 min, 9.46 min, 10.01 min, 10.90 min, 13.34 min, 14.06 min, 16.03 min and 18.98 min, respectively. Excellent linearity was obtained for compounds in the range of concentration: 0.35-42 μg ml⁻¹, 0.5-45 μg ml⁻¹, 4.5-38 μg ml⁻¹, 0.25-25 μg ml⁻¹, 0.5-35 μg ml⁻¹, 0.25-22 μg ml⁻¹, 0.03-52 μg ml⁻¹, 0.15-25 μg ml⁻¹, 0.25-28 μg ml⁻¹, 0.05-18 μg ml⁻¹ and 0.15-35 μg ml⁻¹ for IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET, respectively. The limits of detection (LOD) and limits of quantification (LOQ) for analyzed drugs were calculated in all cases and recovery studies were also performed. Ten human urine samples obtained from patients treated in hospital have been tested. In analyzed samples, one or more drugs from the 11 examined drugs were detected. The concentrations of examined drugs in urine samples ranged between: 1.5-12 μg ml⁻¹ of PAR, 5.2-11.5 μg ml⁻¹ of DPR, 0.13-9.5 μg ml⁻¹ of CFZ and 0.1-8 μg ml⁻¹ of FUR. This method can be successfully applied to routine determination of all these drugs in human urine samples.     

Simultaneous determination of reboxetine and O-desethylreboxetine enantiomers using enantioselective reversed-phase high-performance liquid chromatography

Current knowledge of stereoselective pharmacokinetics and different potencies of drug enantiomers requires the performance of stereoselective analysis during therapeutic drug monitoring in clinical practice. However, in the case of the new antidepressant drug reboxetine, no effort has been made so far to find a such a suitable system. Therefore, as a step towards developing an enantioselective bioanalytical method for reboxetine and the O-desethylreboxetine metabolite, three stereoselective chromatographic approaches have been investigated. Several chiral columns were tested, among them Chiral-AGP, ChiraGrom 2 and Chiral-CBH, which were able to simultaneously separate the two compounds into enantiomers in total running times of 28, 18 and 12 min, respectively.     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Rapid determination of beta-aminoisobutyric acid by reversed-phase high-performance liquid chromatography

For the determination of beta-aminoisobutyric acid (BAIBA) in urine samples in which the beta-alanine concentrations are higher than those of BAIBA, the resolution between these two amino acids, separated by reversed-phase liquid chromatography on an octadecylsilane column, was optimized. The chromatographic analysis included precolumn derivatization of amino acids with o-phthalaldehyde, followed by a 15-min isocratic elution and detection at 340 nm. Because of its simplicity, this method should be useful for monitoring urinary excretion of BAIBA.     

Simultaneous determination of four 5-hydroxy polymethoxyflavones by reversed-phase high performance liquid chromatography with electrochemical detection

Accumulating evidence has suggested the potential health-promoting effects of 5-hydroxy polymethoxyflavones (5-OH-PMFs) naturally existing in citrus genus. However, research efforts are hampered by the lack of reliable and sensitive methods for their determination in plant materials and biological samples. Using reversed-phase high performance liquid chromatography (HPLC) equipped with electrochemical (EC) detection, we have developed a fast and highly sensitive method for quantification of four 5-OH-PMFs, namely 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-6,7,4′-trimethoxyflavone, and 5-hydroxy-6,7,8,4′-tetramethoxyflavone. The method was fully validated in terms of linearity, accuracy and precision. The limit of detection (LOD) was determined as being between 0.65 and 1.8 ng/mL (ppb), demonstrating an over 160 times higher sensitivity in comparison with the previously reported method using UV detection. The recovery rate of the method was between 96.17% and 110.82%, and the precision for the retention times and peak areas was all below 13%. The method was successfully used to quantify 5-OH-PMFs with a wide range of abundance in the citrus products and preparations, such as orange juice, citrus peel, and dried tangerine peel. The quantification method for 5-OH-PMFs developed herein could be useful for the nutritional and pharmacological studies of these compounds in future.     

Faster analysis by reversed-phase high-performance liquid chromatography

Summary Many analysts are not taking full advantage of the high speed possibilities of modern LC. Some analytical procedures reported in the literature, and many in regular use in control laboratories, could be achieved in less time without loss in precision. Some factors which affect retention times are discussed and the advantages and disadvantages of employing shorter column lengths and finer packing materials in reversed-phase HPLC are examined. The effect on efficiency of increased flow rates with 10,5 and 3 m ODS materials is shown. The ability to couple shorter column lengths without loss of efficiency is also demonstrated. This allows a minimum length to be selected that gives adequate resolution. Examples of high speed separations are shown and limitations in state of the art HPLC equipment and chromatographic data systems are discussed briefly.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Simultaneous determination of nine UV filters and four preservatives in suncare products by high-performance liquid chromatography

HPLC method for quantitative determination of four preservatives and nine UV filters worldwide authorized in commercial suncare product was developed and validated, and then 101 samples of commercial suncare products were analyzed for the UV filters and preservatives using the proposed method. The mobile phase was acetonitrile-water containing 0.5% acetic acid using a gradient elution at a flow rate of 0.9 mL/min and UV measurements were carried out at 320 nm for UV filters and 254 nm for preservatives. The correlation coefficients of each calibration curves were mostly higher than 0.999. The percent relative standard deviations (%RSD) ranged from 0.97% to 6.1% for five sample aliquots. The recoveries from the spiked solutions were 98-102%. 2-ethylhexyl-p-methoxycinnamate (EHMC) was detected in 96 of 101 commercial suncare products and the concentration was in the range of 3.08-8.16% and 18 samples were found to exceed the 7.5% which has been defined as the maximum allowed concentration in Korea. Methyl paraben was detected in 81 of 101 samples and the next-most often detected preservatives were propyl paraben (25), ethyl paraben (18), and butyl paraben (4). Three samples of 101 suncare products exceeded the maximum allowed concentration (i.e., 0.58-0.79%). The proposed HPLC method allows efficient and simultaneous analysis of preservatives and UV filters suitable for quality control assays of commercial suncare products.     

Simultaneous determination of metformin in combination with rosiglitazone by reversed-phase liquid chromatography

A simple, rapid, and precise reversed-phase liquid chromatographic method is developed for the simultaneous determination of metformin in combination with rosiglitazone. This method uses a Zorbax XDB C(18) 15-cm analytical column, a mobile phase of acetonitrile and buffer containing 10mM disodium hydrogen phsosphate, and 5mM sodium dodecyl sulphate in the ratio of 34:66 (v/v), and pH is adjusted to 7.1 with orthophosphoric acid. The instrumental settings are a flow rate of 1 mL/min, column temperature at 40 degrees C, and detector wavelength of 226 nm. The internal standard method is used for the quantitation of metformin and rosiglitazone. Methylparaben is used as an internal standard. The method is validated and shown to be linear. The correlation coefficients for metformin and rosiglitazone are 0.9996 and 0.9997, respectively. The relative standard deviation for six replicate measurements in two sets of each drug in the tablets is always less than 2%.     

Simultaneous determination of diclofenac potassium and drotaverine hydrochloride in human plasma using reversed-phase high-performance liquid chromatography

A simple high-performance liquid chromatographic method with ultraviolet detection is proposed for the estimation of diclofenac potassium and drotaverine hydrochloride in human plasma. Liquid-liquid extraction was carried out with a mixture of dichloromethane-isopropyl alcohol (80:20, v/v). Chromatographic separation of the analytes and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed-phase Thermo BDS Hypersil C8 (5 μm particle size) column using a mobile phase of acetonitrile-0.02M ammonium acetate buffer (53:47, v/v) at pH 3.5. The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration ranges of 25-1500 ng/mL and 32-960 ng/mL was obtained for diclofenac potassium and drotaverine hydrochloride, respectively. The limit of quantification was 25 and 32 ng/mL for diclofenac potassium and drotaverine hydrochloride, respectively. Recoveries of diclofenac potassium and drotaverine hydrochloride from plasma were 97.45% and 98.27%, respectively.     

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.