Simultaneous determination of sildenafil and tadalafil in herbal medicines by HPLC

The HPLC method for the simultaneous determination of 2 PDE-5 (sildenafil and tadalafil) in herbal medicines was validated. Using acetonitrile and water as the mobile phase, Gemini C18 (250 x 4.6 nm, 5 µm) column, UV detection at 285 nm and flow at 1 ml/min, the PDE-5 were separated satisfactorily within 6 minutes,

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Journal of military pharmaco-medicine 7-2013

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SIMULTANEOUS DETERMINATION OF SILDENAFIL AND

TADALAFIL IN HERBAL MEDICINES BY HPLC

Nguyen Thi Hao*; Nguyen Van Bach * ; Nguyen Van Thinh * Dao Van Don * ; Pham Van Hinh * ; Nguyen Thi Thanh Phuong **

Summary

The HPLC method for the simultaneous determination of 2 PDE-5 (sildenafil and tadalafil) in herbal medicines was validated Using acetonitrile and water as the mobile phase, Gemini C18 (250 x 4.6 nm, 5 µm) column, UV detection at 285 nm and flow at 1 ml/min, the PDE-5 were separated satisfactorily within 6 minutes The quantitation limits of sildenafil citrate and tadalafil were about 0.2 g/ml and 0.3 g/ml, respectively The calibration curve of each PDE-5 had a correlation coefficient close to 1 The precisions were all less than 2% The recovery rates of extraction were 92 - 100% for all PDE-5 This method can be used to detemine sildenafil citrate and tadalafil simultaneously in herbal medicines

* Key words: PDE-5; Sildenafil citrat; Tadalafil; HPLC; Herbal medicines.

INTRODUCTION

Sildenafil citrate, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4,3-d]pyrimidin-5-yl)phenylsulfonyl]-4 methylpiperazine, is primarily indicated in the treatment of erectile dysfunction It is rapidly absorbed after oral administration, with absolute bioavailability of about 40% It is rapidly and extensively metabolized in the liver to the active N-desmethyl sildenafil metabolite Under steady state conditions, the plasma concentrations of N-desmethyl sildenafil were approximately 40% of those measured for sildenafil

Tadalafil, (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-[3,4-(methylenedioxy) phenyl] pyrazino[1’,2’:1,6]pyrido[3,4-b]indole-1,4-dione [1] is a newly approved oral selective phosphodiesterase-5 (PDE5) inhibitor indicated for the treatment of erectile dysfunction [1, 2, 3, 4] The mechanism of action of tadalafil is similar to the other PDE5 inhibitors, sildenafil Through the inhibition on PDE5, tadalafil increases the concentrations of cyclic guanosine monophosphate (cGMP), producing smooth muscle relaxation and increased blood flow to the corpus cavernosum, thereby enhancing erectile response following appropriate sexual stimulation

Because of its increasing popularity and potential side effects, the need for a procedure to detect both sildenafil and tadalafil in biological samples is becoming increasingly important The simultaneous determination of sildenafil and tadalafil is also necessary for pharmacokinetic and related studies The present work demonstrates that significant improvement in sensitivity was achieved by coupling HPLC with electrochemical detection The simultaneous detection and determination of both analytes was facilitated by the use of two internal standards namely, roxithromycin and clarithromycin, and the use of a one-step liquid–liquid extraction The developed method was validated for its application to a pharmacokinetic evaluation of sildenafil and N-desmethyl sildenafil in 25 healthy human volunteers

MATERIALS AND METHODS

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1 Materials

* Experimental subjects:

- Blank sample: prepared by the extracts of herbals: 10 g of Epimedium grandiflorum; 10 g of Morinda officinalis; 10 g of Cortex eucommiae; 10 g of Semen cuscutae; 10 g of Cibotium barometz; 10 g of Radix dipsaci and 16 g of Herba cistanches

- Test sample: prepare by blank sample added 2 mg/g of SC and TA

* Chemicals and reagents:

- Standards: Sildenaﬁl and tadalaﬁl were purchased from Vietnam National Institute of Drug Control

- Acetonitrile (Merck), water for HPLC

* Analytical equipment:

- HPLC system: HPLC Alliance Waters 2695D system, 4 channel solvent, pump autosampler, column heating chamber, PDA detector, Gemini C18 column (250 x 4,6 mm, 5 µm) Empower ® Software

- Others: Sartorius analytical balance with precision of 0.1 mg, ultrasonic apparatus, glass flasks, precise pipettes

2 Method

* Sample preparation and chromatography:

- Sample preparation: By the results reported [1, 2, 3], preparation procedures were conducted as

follows:

Accurately weight 1 g of sample in 50 ml beaker Extract three times with 10 ml of acetonitrile Ultrasonic shaking for 15 minutes at 300C Filter the extract by filter paper permeated with MeOH into 50 ml flask Combine the extracts into the flask Shake and dilute to volume of flask The solution was filtered by 0,45 m membrane for HPLC injection

- Chromatographic conditions:

Gemini C18 (250 x 4,6 mm, 5 µm) column, PDA detector, Analyses were run at a ﬂow-rate of 1 ml/min, injection volume of 50 µl Investigation of mobile phase was carried out with MeOH/ H2O and ACN /H2O

* Validation of the analytical procedures:

The validations for the following analytical procedures are included: System suitability testing, selectivity - specificity, calibration curve and linearity, precision, accuracy, limit of

detection, lower limit of quantification

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RESULTS AND DISCUSSION

1 Chromatographic conditions

Sample preparation an chromatographic conditions was performed according to 2.2.1 The HPLC method was achieved on a Gemini C18 (250 x 4,6 mm, 5 µm) column, PDA detector; ACN:H2O (60:40, v/v) as mobile phase; flow rate of 1 ml/min; injection volume of 50 µl Fig 1 showed chromatogram of sildenaﬁl and tadalaﬁl

Fig 1: The HPLC chromatograms of sildenaﬁl and tadalaﬁl

2 Validation of the analytical procedures

* System suitability testing:

System suitability testing was examined in six independent samples consisting of SC and

TA at concentraction of 50 µg/ml Results was showned in table 1

Table 1: Results of system suitability testing of SC and TA

No

As can be seen in table 1, the good system suitability was achieved under the chromatographic conditions The relative standard deviation (RSD) of retention time and peak area

t (phút)

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of TA and SC were below 1% showing that the HPLC method can be applied to analyze samples with reliable results

* Selectivity - specificity:

Analyze mixtured standard samples of PR, BE, TR and DA at concentration of 1 µg/ml with

test sample and blank samples Chromatograms were shown in fig 2

Fig 2: Chromatogram of SC and TA in mixed standard sample (a), blank sample (b) and

test sample (c)

TA also displayed appropriate chromatographic retention with its peak sufﬁciently separated from that of SC in the present method The peaks of the analytes in the test sample were symmetrical and completely separate from each other, showing that the HPLC method has good selectivity and specificity

* Calibration curve and linearity:

Prepare a series of standard solutions of PR, BE, TR and DA at concentrations ranging

from 1 - 100 µg/ml and subject to HPLC Results were shown in table 2 and figure 3

Table 2: Results of the linear correlation between concentration and peak area of SC and TA

a

b

c

min

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Speak (TA) (μV.s) 9076 17759 46380 91145 186085 486095 934635

Speak (SC) (μV.s) 28799 57968 105945 268540 546850 1358095 2670189

Calibration curve Equation

y(TA) = 9411x + 268 ; R2 = 0.9996

y(SC) = 26808x - 525 ; R2 = 0.9998

Figure 3: The calibration curve of SC and TA

As can be seen from the table 2, peak area and concentration of PR, BE, TR and DA are

correlated with R2 ≈ 1

* Limit of detection (LOD) and lower limit of quantitation (LOQ):

Based on the baseline noise level of blank and test samples, LOD values of TA và SC

were 0.1 µg/ml; 0.05 µg/ml, respectively LOQ of SC and TA were 0.3 µg/ml and 0.2 µg/ml,

respectively

* Precision:

Weigh accurately 1 g of test sample containing 0.2% of mixed standards Precision was

measured using six determinations at concentration of 0.2% The results were showned in table

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Table 3: Results of precision of the HPLC method (n = 6)

STT

Content of TA

added (%w/w)

Content of TA found (%w/w)

Content of SC added (%w/w)

Content of SC found (%w/w)

b

C (µg/ml)

S (µV.S)

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The relative standard deviations were less than 3%, suggesting that the method have good repeatability

* Accuracy:

Weigh accurately 1 g of the sample with 6 six independent determinations Add 400 µl of mixture of standards at concentration of 1000 µg/ml Tables 4 showed the results of accuracy of the method

Table 4: Precision for the determination of SC and TA

Sample

no

Amount added (µg)

Amount found

TA

X= 95.83%

SD = 1.90

RSD = 1.98%

SC

SD = 1.89

RSD = 1.96%

The mean recovery of TA was 95.83% (n = 6), and that of SC was 96.29% The high recoveries from sample preparation with RSD < 2 indicated that the method offers good

accuracy

CONCLUSION

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A method which employed high-performance liquid chromatography coupled with PDA detector was developed for the simultaneous determination of sildenaﬁl and taladafil The method was developed and validated for purposes of analysis in herbal medicines High precision and accuracy were demonstrated A limit of quantitation (LOQ) of 0.3 µg/ml was achieved for sildenaﬁl whereas a LOQ of 0.3 µg/ml was obtained for tadalafil The mobile phase consisted of ACN:H2O (60:40, v/v) as mobile phase The column was a Gemini C18 (250 x 4.6 mm, 5 µm) column The method is suitable in analysis of sildenafil and tadalafil in herbal medicines

REFERENCES

1 Phan Thanh Mau Study on determination of sidenafil spiked in functional foods (LC-MS HPLC) Pharmacy undergraduate dissertation Hanoi University of Pharmacy 2011

2 Hassan Y Aboul-Enein and Mohamed M Hefnawy RapiddDetermination of sildenaﬁl citrate in pharmaceutical preparations using monolithic silica HPLC column Journal of Liquid Chromatography and

Related Technologies 2003, Vol 26, No 17, pp.2897-2908

3 Samuel R.Gartz PhD E.D drugs and analogs: Characterization of phosphodiesterase-5 Inhibitors in suspect couterfeit and herbal products USP Annual Scientific Meeting 2007

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