Pharmacokinetics and tissue distribution study of 15 ingredients of Polygonum chinense Linn extract in rats by UHPLC-MS/MS.

A rapid and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of 15 bioactive ingredients in rat plasma and tissues after oral administration of Polygonum chinense Linn extract (PCE). After addition of internal standards (ISs; rutin and danshensu), plasma and tissue samples were pre-treated by protein precipitation with acetonitrile-ethanol. The chromatographic separation was performed on an Agilent ZORBAX RRHD Eclipse Plus C18 column with gradient elution using a mobile phase composed of methanol and water (containing 0.2% acetic acid) at a flow rate of 0.3 mL min-1 . Mass spectrometric detection was carried out using a mass spectrometer in both positive and negative ion electrospray ionization modes by multiple reaction monitoring. The method provided excellent linearity, and the lower limit of quantification range 0.5-30 ng mL-1 for all analytes. The intra- and inter-day precision were less than 9.12% and the accuracy ranged from -4.02% to 6.32%, respectively. The mean extraction recovery and matrix effect of analytes and ISs ranged from 83.65% to 109.20%. The method was successfully applied to the pharmacokinetics and tissue distribution study of 15 ingredients of PCE in rats.

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression.

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

An ultra high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of thirteen components extracted from Radix Puerariae in rat plasma and tissues: Application to pharmacokinetic and tissue distribution study.

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p-hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and ß-sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C18 column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol-water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2-35 ng/mL. The intra- and inter-day precision of all the analytes were less than 10.92%, with an accuracy ranging from -13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract.

Simultaneous determination of fourteen compounds of Hedyotis diffusa Willd extract in rats by UHPLC-MS/MS method: Application to pharmacokinetics and tissue distribution study.

A rapid, sensitive and selective ultra high-performance liquid chromatography-tandem mass spectrometry UHPLC-MS/MS method has been developed and validated for the simultaneous determination of fourteen bioactive ingredients (gallic acid, geniposidic acid, protocatechuic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol) in the plasma and tissues of rats. Danshensu and icariin were used as internal standards (IS1 and IS2). The chromatographic separation was achieved by using an Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution using mobile phase, which consisted of 0.1% acetic acid water (solvent A) and methanol (solvent B) and pumped at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode utilizing electrospray ionization (ESI) in positive and negative mode. The plasma samples were pretreated via protein precipitation with 300 µL of methanol containing 0.1% (v/v) formic acid and organ homogenates were processed by solid-phase extraction (SPE) with Waters Oasis HLB 3 cc (60 mg), respectively. The intra- and inter- day precisions (RSD%) were less than 10.3%, while the accuracy was ranged from -7.34% to 9.10%. Extraction recovery ranged from 85.02 to 112.0% and the matrix effects ranged from 85.12% to 109.6%. The present method exhibited excellent linearity and the lower limits of quantification (LLOQ) were 30.0 ng/mL, 15.0 ng/mL, 80.0 ng/mL, 30.0 ng/mL, 10.0 ng/mL, 3.0 ng/mL, 2.5 ng/mL, 2.5 ng/mL, 1.5 ng/mL, 15.0 ng/mL, 75.0 ng/mL, 15.0 ng/mL, 30.0 ng/mL, and 20.0 ng/mL for gallic acid, protocatechuic acid, geniposidic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol, respectively. This analytical method was verified by the FDA guidelines for bioanalytical method validation and applied to investigate the pharmacokinetics and biodistribution of fourteen constituents of Hedyotis diffusa Willd extract in rats. These results provide useful information for improving the pharmacokinetics and biodistribution of fourteen bioactive ingredients of Hedyotis diffusa Willd extract in SD rats, supporting additional clinical application and Chinese herbal medicine safety evaluations.

A UHPLC-MS/MS method for simultaneous determination of twelve constituents from Erigeron breviscapus extract in rat plasma: Application to a pharmacokinetic study.

A rapid, sensitive and specific ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to simultaneously determine the twelve major bioactive ingredients (neochlorogenic acid, chlorogenic acid, caffeic acid, cynarin, scopoletin, scutellarin, isochlorogenic acid A, apigenin-7-o-glucuronide, isochlorogenic acid C, scutellarein, luteolin, and apigenin) in rat plasma. Gallic acid and wogonoside were used as internal standards (IS1 and IS2). The plasma samples were pretreated and extracted by liquid-liquid extraction and protein precipitation with ethyl acetate-acetonitrile (95:5, v/v). Chromatographic separation was accomplished on Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1mm×50mm, 1.8µm) utilizing 0.1% formic acid aqueous solution and acetonitrile as mobile phase under gradient conditions at a flow rate of 0.3mL·min-1. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in positive and negative mode. The whole intra- and inter-day precision (as relative standard deviation) of all analytes were less than 11.03%, and the accuracy (as relative error) were in the range from -10.43% to 9.76% and from -10.14% to 10.33%. The lower limits of quantification (LLOQ) were 20, 3.0, 100, 7.0, 0.30, 2.0, 70, 1.0, 20, 30, 10, and 2.0ngmL-1 for neochlorogenic acid, chlorogenic acid, caffeic acid, cynarin, scopoletin, scutellarin, isochlorogenic acid A, apigenin-7-o-glucuronide, isochlorogenic acid C, scutellarein, luteolin, and apigenin, respectively. Extraction recovery, matrix effect and stability were found to be the required limits. This method was selective and sensitive for the investigation of the pharmacokinetics of twelve constituents following oral administration to research study about in Erigeron breviscapus of clinical practices for separately analytes on rats.

Simultaneous determination and pharmacokinetic study of eight components in rat plasma by UHPLC-MS/MS after oral administration of Hypericum japonicum Thunb extract.

A rapid and sensitive assay based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was established and validated for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in rat plasma using catechin and daidzein as the internal standards (IS). Plasma samples added internal standards were acidified with formic acid then pretreated by direct protein precipitation with acetonitrile. The separation of eight constituents was achieved on a C18 column with gradient elution using methanol and 0.2% acetic acid aqueous solution as the mobile phase and detected by multiple reaction monitoring using electrospray ionization source in the positive-negative ionization mode. The method was validated for sufficient specificity, precision, accuracy, and sensitivity over the concentration range of 10-6000 ng mL(-1) for gallic acid, 1.5-3000 ng mL(-1) for protocatechuic acid, 10-15000 ng mL(-1) for vanillic acid, 2-3600 ng mL(-1) for caffeic acid, 1.5-3600 ng mL(-1) for epicatechin, 4-6000 ng mL(-1) for isoquercitrin, 2-9000 ng mL(-1) for vincetoxicoside B, and 20-18000 ng mL(-1) for quercetin. The overall intra­run precision and the inter­run precision were showed in the range of 1.0-14.2% and 2.8-12.9%, respectively, and the accuracy was no more than 12.8%. This analytical method was successfully applied to investigate the pharmacokinetics of eight ingredients in rats after oral administration of Hypericum japonicum Thunb extract.

The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 µM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways.

Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner.

OBJECTIVES: Icariin, a flavonoid isolated from Epimedium pubescens, has previously been identified to exert beneficial effects on preventing bone loss and promoting bone regeneration. However, molecular mechanisms for its anabolic action have, up to now, remained largely unknown. MATERIALS AND METHODS: Effects of icariin on cell proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) were systematically evaluated. To characterize underlying mechanisms, its effects on mitogen-activated protein kinase (MAPK) signalling pathways were determined. RESULTS: Results showed that icariin might not have enhanced effects on cell proliferation. However, it seemed to significantly enhance osteogenic differentiation of BMSCs, demonstrated by increasing alkaline phosphatase (ALP) activity and gene expression of collagen type I (Col I), osteocalcin (OCN) and osteopotin (OPN). It was demonstrated that icariin rapidly phosphorylated extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun N terminal kinase (JNK). Furthermore, icariin-stimulated osteogenic effects on BMSCs were dramatically attenuated by treatment with either specific ERK inhibitor of PD98059, p38 inhibitor of SB202190 or JNK inhibitor SP600125. CONCLUSIONS: These results provide a potential mechanism of anabolic activity of icariin on BMSCs involving ERK, p38 and JNK MAPK pathways.

Simultaneous determination of ten active constituents of Yankening Capsule in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry.

An ultra high performance liquid chromatography with tandem mass spectrometry (U-HPLC-MS/MS) method was developed for simultaneous determination and pharmacokinetic study of ten active constituents, phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin in rat plasma after oral administration of Yankening Capsule. After mixing with two internal standards tetrahydropalmatine and rutin, plasma samples were pretreated by protein precipitation with anhydrous ethanol-acetonitrile (9:1, v/v). The U-HPLC separation was carried on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution using a mobile phase composed of methanol and water (containing 0.3% formic acid) at a flow rate of 0.3 mL min(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive-negative ionization mode. The calibration curves of ten analytes showed good linearity (r>0.9979). The lower limits of quantification of phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin were 0.50, 0.50, 0.30, 0.30, 0.30, 10, 3.0, 8.0, 1.0, 8.0 µg L(-1), respectively. The relative standard deviation of intra-day precision and inter-day precision were in the range from 1.13% to 5.96% and from 0.65% to 8.85%, respectively. The matrix effects of all analytes were found to be within the acceptable range with a range of 89.99-109.3%. The method is reliable and rapid and has been applied successfully to pharmacokinetic study of the ten active constituents in rat plasma after oral administration of Yankening Capsule.

[Simultaneous determination of ten constituents in the Chinese medicinal preparation fufangxingxiangtu' erfeng capsules by ultra performance liquid chromatography with tandem mass spectrometry].

Using caffeic acid and icariin as internal standards, a method for the simultaneous determination of protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, scutellarin, isochlorogenic acid C, baicalin, luteolin, apigenin, atractylenolide III and atractylenolide I in Fufangxingxiangtu' erfeng capsules were established by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The separation was performed on a ZORBAX RRHD Eclipse Plus C18 column (50 mm x 2.1 mm, 1.8 microm) by using water containing 0.3% formic acid and methanol as mobile phases with the gradient elution at a flow rate of 0.3 mL/min. The analytes were detected by a tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via the switching of positive electrospray ionization (ESI(+)) and negative electrospray ionization (ESI(-)). Under optimum conditions, the calibration curves were linear in the range of 0.003 00-24.0 mg/L for protocatechuic acid, 0.017 0-2.00 mg/L for protocatechuic aldehyde, 0.015 0-30.0 mg/L for chlorogenic acid, 0.004 00-30.0 mg/L for scutellarin, 0.010 5-24.0 mg/L for isochlorogenic acid C, 0.003 00-30.0 mg/L for baicalin, 0.003 00-5.0 mg/L for luteolin, 0.006 00-1.50 mg/L for apigenin, 0.001 50-4.00 mg/L for atractylenolide III, and 0.000 600-0.900 mg/L for atractylenolide I with the detection limits of 1.0, 11, 5.0, 1.5, 3.5, 1.0, 1.0, 2.0, 0.50, 0.20 microg/L, respectively. The average recoveries of the ten effective components were between 92.5% and 106% with all relative standard deviations not more than 3.2%. The developed method was rapid, simple, accurate, reproducible, and suitable for the quality control of the Fufangxingxiangtu' erfeng capsules.

[Simultaneous determination of five alkaloids in Niuhuang Shangqing Tablets by micellar electrokinetic chromatography-mass spectrometry using laurel acyl malic acid ester].

A micellar electrokinetic chromatography-mass spectrometric method based on laurel acyl malic acid ester (LMAE) for the separation and determination of coptisine, berberine, jatrorrhizine, phellodendrine and ligustrazine in Niuhuang Shangqing Tablets was established. The baseline separation of the five compounds was attained within 18 min by an uncoated capillary (88 cm x 50 microm) on the operating voltage of 25 kV using 7.5 mmol/L LMAE-15 mmol/L ammonia-50 mmol/L ammonium acetate mixture (pH = 7.0) containing 12.5% (v/v) acetonitrile as the electrophoretic medium and 50% 2-propanol aqueous solution (containing 3 mmol/L acetic acid) as the sheath liquid. The peak area of each component to its concentration showed a good linear relationship. The relative standard deviations of migration times and peak areas of the five components were less than 5% and the recoveries were between 96.0% and 105%. The developed method is simple, rapid, accurate and is suitable for the routine analysis of the five alkaloid components in Niuhuang Shangqing Tablets.

[Simultaneous determination of nine effective components in jiaweizuojin pills by ultra performance liquid chromatography with tandem mass spectrometry].

A method for the simultaneous determination of paeoniflorin, tetrahydropalmatine, jatrorrhizine, berberine, palmatine, evodiamine, saikosaponin C, saikosaponin A and saikosaponin D in Jiaweizuojin Pills was established by ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The UPLC separation was performed on a Zorbax RRHD Eclipse Plus C18 column (50 mm x 2.1 mm, 1.8 microm) by using 0.2% (v/v) formic acid aqueous solution and methanol as mobile phases with the gradient elution at a flow rate of 0.4 mL/min. The analytes were detected by tandem mass spectrometry under the positive ion mode with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. Under the optimized conditions, the calibration curves were linear in the ranges of 0.025 -5.0 mg/L for paeoniflorin, 0.0010-2.0 mg/L for tetrahydropalmatine, 0.0023-7.2 mg/L for jatrorrhizine, 0.0027-28.9 mg/L for berberine, 0. 002 3 - 9. 1 mg/L for palmatine, 0. 005 0 -1.0 mg/L for evodiamine, 0.050-10 mg/L for saikosaponin C, 0.0050-1.0 mg/L for saikosaponin A and 0.0075-1.5 mg/L for saikosaponin D with the detection limits of 5.0, 0.20, 0.45, 0.54, 0.45, 1.0, 10, 1.0, 1.5 microg/L, respectively. The average recoveries of the nine effective components were between 99.3% and 105% with the relative standard deviations not more than 2.6%. The developed method is simple, rapid and accurate, and suitable for the quality control of the nine effective components in jiaweizuojin pills.

[Simultaneous determination of berberine, palmatine, matrine, catechin and baicalin in Funing Shuan by micellar electrokinetic capillary chromatography-electrospray ionization mass spectrometry].

A method for the simultaneous determination of berberine, palmatine, matrine, catechin and baicalin in Funing Shuan was established using micellar electrokinetic capillary chromatography-electrospray ionization mass spectrometry (MEKC-ESI MS). The compounds were separated on an uncoated capillary (80 cm x 50 microm) with the operating voltage of 25 kV and the running buffer of 40 mmol/L lauric acid-100 mmol/L ammonia mixture containing 25% acetonitrile (pH 9.5). The baseline separation of the five compounds was achieved within 16 min with a satisfactory repeatability and sensitivity. The solution of 50% 2-propanol/water solution (containing 3 mmol/L acetic acid) was used as the sheath liquid for the ESI MS analysis. The results showed that the linear ranges for berberine, palmatine, matrine, catechin and baicalin were 0.03 - 15, 0.05 - 15, 0.2 - 250, 1.5 - 300 and 2.0 - 500 mg/L, respectively, and the detection limits were 0.01, 0.02, 0.05, 0.5 and 0.6 mg/L, respectively. The average recoveries of the five components were between 94.0% - 104.0% with the relative standard deviations (RSDs) of 0.3% - 3.2%. The developed method is simple, rapid, and accurate, and it is suitable for the routine analysis of the five effective components in Funing Shuan.

[Determination of organic acids in cane vinasse by micellar electrokinetic capillary chromatography with indirect ultraviolet detection].

Micellar electrokinetic capillary chromatography (MECC) with indirect ultraviolet (UV) detection method for the separation and determination of several organic acids in cane vinasse, including malonic, formic, tartaric, malic, succinic, glutaric, acetic, lactic and glutamic acids, were developed. Electrophoretic conditions were as follows: uncoated fused silica capillary (56 cm/ 64 cm (effective/total length), 50 microm i. d. ), 7.5 mmol/L potassium acid phthalate-1. 5 mmol/L cetyltrimethyl-ammonium bromide (CTAB) at pH = 6.50 as buffer solution, applied voltage -25 kV, temperature 25 degrees C, detection wavelength 300 nm, reference wavelength 210 nm. Good linearities were obtained for nine organic acids, and the detection limits were 0.5 mg/L, 0.3 mg/L, 1.5 mg/L, 1.5 mg/L, 0.3 mg/L, 0.3 mg/L, 0.4 mg/L, 0.4 mg/L, 0.4 mg/L for malonic, formic, tartaric, malic, succinic, glutaric, acetic, lactic and glutamic acid, respectively. The relative standard deviations (RSDs) for migration times and peak areas of nine organic acids within a day were 0.4% - 0.6% and 2.3% - 4.8%, respectively. The corresponding data for five days were 0.5% -0.7% and 3.3% - 5.2%. The recoveries of acid standards were above 93%. The method can be applied to determine the organic acids in cane vinasse with satisfactory results.

Semi-industrial isolation of salicin and amygdalin from plant extracts using slow rotary counter-current chromatography.

Salicin in the bark extract of Salix alba and amygdalin in the fruit extract of Semen armeniacae were each separated by slow rotary counter-current chromatography (SRCCC). The apparatus was equipped with a 40-L column made of 17 mm i.d. convoluted Teflon tubing. A 500g amount of crude extract containing salicin at 13.5% was separated yielding 63.5 g of salicin at 95.3% purity in 20h using methyl tert-butyl ether-l-butanol (1:3) saturated by methanol-water (1:5) as a stationary phase and methanol-water (1:5) saturated by methyl tert-butyl ether-1-butanol (1:3) as a mobile phase. A 400g amount of crude extract containing amygdalin at 55.3% was isolated to yield 221.2g of amygdalin at 94.1% purity in 19h using ethyl acetate-1-butanol (1:2) saturated by water as a stationary phase and water saturated by ethyl acetate-1-butanol (1:2) as a mobile phase. The flow rate of the mobile phase was 50 ml/min. The results show that industrial SRCCC separation of salicin and amygdalin is feasible using a larger column at a higher flow rate of the mobile phase.

[Determination of apomorphine, sildenafil and alprostadil in medicines for erectile dysfunction by high performance liquid chromatography-mass spectrometry].

A high performance liquid chromatography-mass spectrometry (LC-MS) analytical method for illicit drugs, apomorphine, sildenafil and alprostadil, in medicines for erectile dysfunction has been developed. The samples were extracted with methanol using ultrasound-assisted extraction. The chromatographic separation was performed on a Zorbax Eclipse XDB-C18 column using acetonitrile-0.5% formic acid aqueous solution as mobile phase. The three compounds were identified by retention time and m/z and quantified by peak area. The results demonstrated that the linear ranges were 50.0 - 5 000.0 microg/L, 10.0 - 1 000.0 microg/L, 40.0 - 4 000.0 microg/L, with detection limits of 20.0, 4.0, 10.0 microg/L for apomorphine, sildenafil and alprostadil, respectively. The average recoveries and the relative standard deviations were 89% - 95% and 9.5% - 11%. The method is simple, rapid, accurate and suitable for the simultaneous determination of these drugs in medicines for erectile dysfunction.