Indian Journal of Chemistry Vol. 44A, April 2005, pp. 737-740

Spectrophotometric method for the simulta­neous determination of piroxicam and

2-aminopyridine

L Suntornsuk*, P Bunavitayakij & P Pitayati enanan Departm; nt of Pharmaceutical Chemistry, Faculty of Pharmacy,

Mahidol University, 447 Sri-Ayudhaya Re. , Rajathevee, Bangkok 10400 .... T hai land

Email : py lll @mah idol.ac .th

Received 30 Septelllber 2004; revised 20 Jal/uary 2005

A rapid and simple spectrophotometri c method is proposed for the simultaneous determ inati on of pirox icam (Pi) and 2-aminopyridine (2-Ap). Pirox icam is stable under bas ic hydro lysis, but y ields 2-Ap as one of the degradati on products under ac id hydrolysis. The method is based on the measurement o f absorban­ces o f 2-Ap and Pi at 300 and 360 nm. respectively, and the cal­cu lations are based on the binary method. The absorbances of both compounds obey Beer-Lambert 's law over the concentration

ran ge of 5-25 ~lg L ' ! wi th good lineari ty (/>0.99). The recoveries are with in 100.8- 106.4% for Pi and are within 96.4-98.9% for 2-Ap. Prec ision is good wi th acceptable limits of detect ion (LOD) and quantitation (LOQ) for both compounds. The method has been app lied for the determination o f Pi and 2-Ap in pi roxicam capsules. The average content of two di fferent brands o f pirox i ­cam is 97.4 and 98.5% (n = 3), wh ich comp lies wi th the USP 26 (92.5- 107.5%). Under the stress conditi on (renuxi ng w ith 0. 1 N HCI). the percentages of pi rox icam decrease from 100% (0 h) to 18.9% (2 1 h) and 2-Ap increase from 0% (0 h) to 63.6% (2 1 h).

IPC Code: Int. Cl7 GOI 21100

Pirox icam (Pi), 4-hydroxy-2-methy-N-(2-py ridyl)-2H-I ,2-benzothiazine-3-carboxamide-l , I-d iox ide, is a prototype of a non-steroidal anti-innam matory drug ( ISAID). The drug is an enolic acid, whi ch exis ts in two tautomeri c forms l

, with a pKa of 6.3 in dioxane:water (2: 1) 1.2. Piroxicam is inso luble in water with a partition coefficient of 1.8 between octanol and aq ueous buffer (p H 7.4)'.

The synthet ic pathways of pi rox icam are al ready known and the final step in the sy nthetic route involves the treatment of compound 5 with 2-aminopy ri di ne (2-Ap) in reflu xing xy lene3. 2-Ap is not on ly used as a reagent in the sy nthesis, but it is also one of the degradation products of piroxicam. The presence of the 2-Ap residue in pi rox icam raw material or as the degraded compound in piroxicam fo rmu lati ons is undes irab le. 2-Ap can cause the irritation of eyes, nose, throat, headache, dizz iness, exc itement , nausea, hi gh blood pressure. respiratory

di stress, weakness, convulsion and stupor4• Toxicity

data in human show that after 5 h exposure to 5 lAg mrl of 2-Ap causes severe headache, increased blood pressure, flu shing of the extremities and nausea5.

Several analytical methods have been reported fo r the analys is of piroxicam in formulations such as spectrophotometrl, spectronuorometry 7.S, high­performance liquid chromatography (HPLC)9-1 1 and capillary electrophoresis (CE)12 . Determination of the drug in human plasma and urine has been demonstrated mainly by HPLC I3,15. Stability indicating assays of piroxicam have been compared by HPLC, CE and high-performance thin layer chromatography (HPTLC) '6. '7,

A rapid and simple spectrophotometric method for the analysis of pi rox icam and 2-Ap and stud ies on the stability of piroxicam under acid and basic hydrolys is are reported here. The remaining piroxicam and the generated 2-A p are calculated using the binary method IS. The method is va luab le where a mixture shows overlapped ultra-violet (UV) spectra, i.e., the absorbance of compound X is interfered by that of co mpound Y and vice versa. Thus, the absorbance of each compound is the sum of the absorbance of both compounds present in the mixture as show n in Eqs ( I) and (2). The amount of the individual compound can be deri ved by Eqs (3) and (4), which are obtained by the substitution of Eqs ( I) and (2) with Beer­Lambert 's law (A = ahc).

A AX = Ax),x + AyAx

A),y = A xAy + A yAy

A Ax = axAXhcX + a y),xhcy

A AY = a xAyhcx + ClyAybcy

( I ) (2) (3)

(4)

where, A ),x is the absorbance of the mi xture at the maximum wavelength of compound X; A i,y the absorbance of the mi xture at the max imum wavelength of compound Y; A x. A y the absorbance of co mpound X and Y, respecti vely; Ax, Ay the maxi mum wavelength of compound X and Y, respectively; a absorpti vity (L g - I cm' I); h path length (cm) : and , c the concentration (g L'I).

Experimental Ana lyti cal grade reagents and solvents were used

here. Piroxicam and 2-am inopyridine were obtained

738 INDIAN J CHEM SEC A, APRIL 2005

from Sigma (St. Louis, MO, USA). Piroxicam capsules were from SeaPharm Manufacturing (Ayutthaya, Thailand). Hydrochloric acid was obtained from BHD Lab (Poole, England) and sodium hydroxide was from LabGuard (Xalostoc, Mexico). Double distilled water was used.

Stock standard solutions of piroxicam and 2-Ap (10 mg L") was prepared by separately dissolving appropriate amount of each compound in 0.1 N HCI. Working standard solutions were obtained by diluting the stock standard solution with water to obtain the concentration of 1 mg L-' and adjusted pH to 7.0 with 0.1 N NaOH. The standard mixture solutions were prepared by mixing the working standard solutions to obtain the concentration of piroxicam and 2-Ap at ratios of 10:0, 7:3, 5:5, 3:7 and 0: 10 ~Lg L-'. Solutions for calibration data were obtained from diluting the working standard solution to the final concentration of 5-25 ~g L-'.

pH measurements were carried out with a Consort C830 pH meter equipped with a glass combined electrode (Turnhout, Belgium). UV absorbance and spectra were obtained from a UV-160A Shidmadzu.

Assay of piroxicam in capsules by spectrophotometry

Solvents and sonication time were optimized using standard piroxicam. Ten mg of piroxicam was di ssolved in a mixture of water and methanol at vari­ous ratios (100:0, 75 :25. 50:50, 25:75 and 0: 100 v/v) and sonicated in an ultrasonic bath for different times (0, [0, [5 , 20 and 25 min). The optimum condition was employed for establishment of a calibration curve in a range of 5-25 ~g L-' and UV absorbance was ob­tained by measurement at the maximum wavelength scanning in a range of 200-600 nm.

Powder from twenty capsules of piroxicam cap­sules was mixed and finely grounded. An amount equivalent to 10 mg of the drug was weighed accu­rately , transferred into a 100 mL volumetric flask anti diluted with the appropriate solvent. The solution was sonicated, filtered through a Whatman paper No. I and diluted to obtain the final concentration of [0 ~g L-'. The solution was analyzed as described earlier.

Stability study of piroxicam under acid and basic hydrolysis A preliminary study on the stability of piroxicam

under acid and basic hydrolysis of piroxicam was per­formed by refluxing 10 mg of the drug in [00 mL of 0.1 N hydrochloric acid or 0.1 N sodium hydroxide at 100°C. Aliquots were taken after 0, 3 and 6 h, divided

into two portions and each was diluted to obtain the concentration of 10 ~g L-'of the original concen­tration . One portion was neutralized to pH 7.0 with 0.1 N hydrochloric acid or 0.1 N sodium hydroxide and the others were kept un-neutralized at pH 1 or pH 13, respectively. UV spectra of all solutions were scanned in a range of 200-600 nm. The conditions, which gave 2-Ap as the degradation product was fur­ther employed for the stability study of piroxicam capsules.

Analytical performance characteristics

Analytical performance characteristics including linearity, precision, accuracy, limit of detection and limit of quantitation for the analyses of 2-Ap and the remaining piroxicam and by spectrophotometry using the binary method calculation were evaluated. Cali­bration curves were obtained by measurement the UV absorbance of the standard solutions of piroxicam and 2-Ap in a range of 5-25 ~g L-' . Linear regression and correlation coefficient (r2) were calculated using Mi­crosoft Excel® program. Precision of the method was determined by repetitive measurements (n = 5) of the UV absorbance of the pure piroxicam and 2-Ap solu­tions and percent relative standard deviations (%RSD) were calculated. Recoveries (%R) of the method were determined by preparing synthetic mixtures contain­ing piroxicam and 2-Ap at ratios of 7:3, 5:5 and 3:7 Ilg L-' and %R was calculated by the ratios of the amount added and amount found. Limi ts of detection (LOD) and quantitation (LOQ) were calculated from 3.3 and 10 times of the ratios of the standard deviation and the slope of the calibration curve, respectively .

Results and discussion Assay of piroxicam in capsules by spectrophotometry

Assay of piroxicam in capsules was performed to check whether the piroxicam content in the investi­gated capsules complied with the USP 26 require­ment9

. The optimal solvent for di ssolvi ng piroxicam was in 50% v/v methanol and the appropriate sonica­tion time was 15 min . The maximum UV absorbance of piroxicam was obtained at the wavelength of 360 nm and the linear equation was y = 0.0522x + 0.0112 (/ = 0.9997). The average content of two different brands of piroxicam was 97.4 and 98.5% (n = 3), which complies with the USP 26 (92.5-107 .5%).

Stability study of piroxicam under acid and basic hydrolysis

Results from preliminary study on the stability of piroxicam under acid and basic hydrolysis show that

NOTES 739

120

100

[ 80

S 60 0 E ..: 40

20

0

0 10 20 30

Rcllux timc (h)

Fig. I- Percentagcs of piroxicalll ( . ) and 2-alll inopyridine (D ) after relluxing in 0. 1 N HCI

Tab le I--Opt ical characteri stics, linearity and sensiti vity of the compounds

Parameter Piroxicam 2-A mino-pyridine

An"" (nm) 360 300 Beer-Lambert' s l:.Jw limit (~tg mL' I) 5-25 7-35 Absorpli l' ily (I mol'l cm' l) :1 t 300 nm 7.239 6.2 12 at 360 nm 2 1.3 15 32 S:1nclell's sensitivity 5.6 x la" 7.3 x 10" (pg cm,l per 0.00 I absorbance unit) Correlation coefficient (/) 0.9966 0.999 1 Regressioll eqll lllioll (.1')

Slope. ({ 0.0536 0.06·B %RSD of slope (n = 5) 0.1 6 0.00 Intercep t. b 0.On7 0.0389 '7o RSD of in tercept (n = 5) 0.84 0.66 Limit of detection ( ~tg mL' I) 6.3 4.7

Limit of quantitation (pg mL' I) 19.0 14.3

UV absorbance o f pirox icam at 360 nm re mained unchanged afte r refluxin g in basic condition fo r 3 o r 6 h, but in ac id conditi o n the value drama ti ca ll y decreased . Percents o f 2-Ap and the re ma ining piroxicam ,vere calcula ted by the binary method l 8

.

The percen tages o r piroxic<tm were unaflcc ted unde r basic hydrolysis, which were 100% and 99.0% after 0 and 6 h, respectively. However, the percentages o f piroxicam decreased from 100% (0 h) to 43 .6 % (6 h) under ac id hydro lys is . We reasoned that ac id hydro lys is o f piroxicam easily occ urred at th e higher rate than basic hydro lys is due to the protonation of the amine group of the pyri di ne ring . The hypsochromi c shift (blue hift) of pirox ica m fro m 360 to 332 nm occurred when the p H of the so luti o n changed fro m

pH 7 (neutralized soluti on) to pH I (non-neutralized solution) , respectively. Additionally , the UV spectrum from acid hydrolysis of the neutra lized solution (pH=7) shows the second peak of the degraded product at about 300 nm, which was sharper than that obtained from the non-neutralized solution (pH=l). The identity of the degradation product, 2-Ap, was confirmed by comparison of its UV spectrum with that of the standard 2-Ap, which provided the same peak shape and the same maximum absorbance at the wavelength of 300 nm. It was concluded that piroxicam was stable under basic hydrolysis, whereas it degraded under acid hydrolysis and yie lded 2-Ap as one of the degradation products.

Further study foc used o n the ac id hydrolysi s o f pirox icam from capsu les by reflu xi ng the drug fo r a lo nger period . In an attempt to ob tain the complete ac id hydrol ysis, the refl uxing time was increased from 0-2 1 h. Aliquots were prepared as described earli er, the UV absorbance was measured at 300 and 360 nm and percentages of the re mained piroxicam and 2-Ap were calcul ated by the binary method. Fi g. I shows that, after reflux ing in 0 . 1 N hydroch lori c acid , the pe rcentages of piroxicam decreased from 100% (0 h) to 18.9% (2 1 h) and 2-Ap increased from 0% (0 h) to 63.6% (2 1 h). In co mpari son, the sa me lot of pirox icam capsules, whi ch were not reflu xed wi th hydrochlo ri c acid , and the 2-Ap spiked samples were ana lyzed . These sampl es were disso lved in 0.1 N Hel , sonicated for 30 min , filte red and di lu ted to the desirable concentrat ions . This conditi on did not cause any degradati o n of piroxicam . UV absorbance of all samp les were measured as described earli er. Data showed that the sampl es did not contain any

significant amoun t of 2-Ap (LOD = 4.7 ~tg L' I).

Analytical performance characteristics

The va li dity of the methods for the analyses of pi rox ica m and 2-Ap was exa mined usi ng the proposed procedures. Summary o f optical and analytical performance characteri sti cs is shown in T ab le I. The ca libratio n curves and linear equations o f pi rox icam at the wavelengths of 300 and 360 nm were y = O.022 lx - 0 .0099 (/ = 0 .9999) and v = 0 .S36x + 0.0737 (/ = 0.9966), respecti vely. 'For 2-Ap, the linear eq uati o n at the wavelengths o f 300 was y = 0.0643x + 0 .0389 (/ = 0 .999 1). At the wavelength o f 360 nm. 2-Ap d id not show sig ni ficalll absorpt io n. From the absorptivity va lues (Tab le I), and calibrati o n curves, it is ev ident that piroxicam strongly inte rferes the UV abso rbance of 2-Ap at the wa e length of 300 11 m.

740 INDIAN J CHEM SEC A, APRIL 2005

Whereas, 2-Ap. did not interfere the UV absorbance of piroxicam at the wavelength of 360 nm. This is a situation when the binary method plays an important role for calculations. Precision of the method, calculated from %RSDs of the absorbance of piroxicam and 2-AP at the wavelengths of 300 and 360 nm, were less than 5.23% for all cases (n = 5) and recoveries of piroxicam were within 100.8-106.4% (%RSD = 0.04%) and of 2-AP were within 96.4-98 .9% (%RSD = 0.03). The LOD and LOQ values for both compounds were within 6.3 and 19.0 ).!g mr', respectively (Table I).

Conclusion Unlike other researchers, which use advanced

techniques (e.g. HPLC, HPTLC and CE), we describe the utilization of spectrophotometric method for the determination of piroxicam and its degradation product, 2-Ap. Additionally, the direct UV measure­ment is rapid, convenient and in-expensive.

Acknowledgement Sincere thanks are due to the Thailand Research

Fund (TRF) for providing the financjal . support (IRPUS-FS0003/46), the Faculty of Pharmacy , Mahidol Universi ty for providing the facilities, Ms Lawan Satrabuddha from SeaPharm Manufact!]ring for providing piroxicam standard and samples. Thanks are also due to Prof. August Wilhelm Frahm, Department of Pharmaceutical Chemistry, University of Frieburg and Prof. Wolfgang Buchberger, Institute of Analytical Chemistry, University of Linz for their valuable suggestions.

References Roberts II J L & Morrow J D, in The Pharmacological Basis of Therapeutics, edi ted by J G Hardman , L E Limbird,

Goodman & Gilman (Mc-Graw-Hill , New York), 2001 , pp. 713-714.

2 Paulus H E, Furst D E & Dromgool S H, Drugs for Rheu­matic Disease (Churchill Livingston, New York), 1987.

3 Mihalic M, Hofman H, Kuftinec J, Krile B, Caplar V, Kajfez F & Blazevic N, in Analytical Profiles of Drug Substances, edited by K Florey, A A AI-Badr, G A Brewer (Jr), G S Brenner, N J DeAngeli s & J A Mollica (Academic Press, Inc., Tokyo), 1986, pp. 509-531.

4 Budavari S, O'Neil M J, Smith A & Heckelman P E, in The Merck Index: An Encyclopedia of Chemicals. Drugs. alld Biologicals (Merck & Co., Inc. Whitehouse Station; New Jersey) , 2001, pp. 457 .

5 Watrous R M & Schuz H N, Ind Med Surg. 19 (1950) 317-320.

6 Nagaralli B S, Seetharamappa J & Melwanki M B, J Pharm Biomed Anal. 29 (2002) 859-864.

7 Escandar G M, Bystol A J & Campiglia A D, Allal Chim Acta, 466 (2002) 275-283.

8 · Damiani P C, Bearzotti M, Cabezon M & Olivieri A C, J Pharm Biomed Allal. 17 (1998) 233-236.

9 United States Pharmacopoeia 26. National Formulary 21, (The United States Pharmacopeial Convention, Inc., Rockville), 2003 , pp. 1486-1487.

10 Owen S G, Roberts M S & Friesen W T , J Chroll1atogr B. 416 ( 1987) 293-302.

I I British Pharmacopoeia (Stationery office, London), 200 I, pp.1320-1322.

12 Donato M G, Baeyens W, Van Der Bossche W & Sandra P, J Pharm BioI/led Anal. 12 ( 1994) 21-26.

13 Hirai T, Matsumoto S & Ki shi I, J Chromatogr B. 692 (1997) 375-388.

14 Twomey T M, Bartolucci S R & Hobbs D C, J Chromatogr B, 183 ( 1980) 104-108.

15 Richardson C J, Ross S G, Blocka K L & Verbeeck R K. J Chroll1atogr B. 382 (1986) 382-388.

16 Bartsch H, Eiper A & Kopelent-Frank H. Stability indicating assays for the determinatio n of piroxicam-compar ison o f methods, J Pharm Bioll1ed Anal. 20 ( 1999) 53 1-541.

17 Puthli S P & Vavia P R, J Pharlll BioI/l u i Allal. 22 (2000) 673-677.

18 Fritz J S & Schenk G H, Quantitative Analytical Chemist,y, (Bacon Inc., New York), 1987, pp. 360.