CHEM. RES. CHINESE UNIVERSITIES 2009, 25(4),

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1 CHEM. RES. CHINESE UNIVERSITIES 2009, 25(4), Quantitative Determination of Adrenaline Hydrochloride Injection and Noradrenaline Bitartrate Injection by a New HPLC Method via Substitute for Reference XIE Yuan-chao 1,2*, HUANG Hai-wei 3, ZHANG Qi-ming 3 and JIN Shao-hong 3 1. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing , P. R. China; 2. Shandong Provincial Institute for Drug Control, Jinan , P. R. China; 3. National Institute for Control of Pharmaceutical and Biological Products, Beijing , P. R. China Abstract An HPLC method for quantitative determination of adrenaline hydrochloride injection and noradrenaline bitartrate injection was established and validated with a substitute for the reference substance. Phenylephrine hydrochloride was selected as the substitute for the reference substance of adrenaline and noradrenaline bitartrate. The correction factor of phenylephrine hydrochloride with respect to the reference substance of adrenaline and noradrenaline bitartrate was determined under defined conditions. Adrenaline hydrochloride injection and noradrenaline bitartrate injection were quantified by assaying phenylephrine hydrochloride and a correct factor. The results indicate that the HPLC method with the substitute for reference substance was reliable and feasible for quantitative determination of drugs. Keywords Substitute for reference substance; Correction factor; Adrenaline; Noradrenaline bitartrate; Phenylephrine hydrochloride; HPLC Article ID (2009) Introduction Reference substance is crucial for the determination of composition of drugs in quality control. However, in practice, some of the reference substances are relatively expensive or unstable in nature, which are hard to fully obtain, consequently, it is infeasible to ascertain several crucial indices of those drugs in the process of quality control. Therefore, it is very imperative to develop a substitute analytical method for selecting an obtainable, cheap and stable substance named the substitute for reference substance to supersede the original reference substance in drugs assaying. In European Pharmacopoeia 5.0, 1 mg of butylparahydroxybenzoate corresponds to 8.65 mg of allicin to calculate the percentage of allicin [1]. The relative correction factor( f ) of the substitute for reference substance with respect to the reference substance was assayed firstly and then the content of drugs were determined quantitatively by using the substitute for reference substance combined with the correction factor. The formula of f is f = A i/ ci Ar/ cr *Corresponding author. sdxyc@yahoo.com.cn Received March 10, 2009; accepted March 17, 2009., in which A i is the peak area of the substitute for reference substance, A r is the peak area of reference substance, c i is the concentration of the substitute for reference substance and c r is the concentration of reference substance. In order to determine the content for drugs using the substitute for reference substance, the f value should be detected. A series of standard solutions of reference substance and stustitute for reference substance with different concentrations was prepared under certain chromatographic conditions with the concentration as X-axis and the peak area as Y-axis. The calibration curves of the reference substance and its substitute were then determined. After the intercepts of the calibration curves were adjusted to 0, the correction factor( f ) was calculated with the ratio of the two rates of the standard curves(k) [2]. When the sample was analyzed, the concentration c= f At A/c i i when using the substitute for reference substance, in which A t is the peak area of the detected sample. High-performance liquid chromatography(hplc) method has been developed for the determination of Adrenaline hydrochloride injection [3] and noradrenaline bitartrate injection [4] in CP 2005(Chinese

2 434 CHEM. RES. CHINESE UNIVERSITIES Vol.25 Pharmacopeia Second Portion). However, the reference substance of adrenaline and noradrenaline bitartrate is relatively unstable, thus hinders the practical performance of quality control of drugs. This paper presents a new analytical method of using a substitute phenylephrine hydrochloride reference substance for adrenaline and noradrenaline bitartrate reference substance to quantitatively determine the content of adrenaline and noradrenaline bitartrate injection on HPLC, which validates its feasibility in the process of drug quality control analysis. 2 Experimental 2.1 Chemicals and Reagents Adrenaline reference substance(purity 100.0%, Batch No ), noradrenaline bitartrate reference substance(purity 100.0%, Batch No ) and phenylephrine hydrochloride reference substance(purity 100.0%, Batch No ) were provided by National Institute for Control of Pharmaceutical and Biological Products(Beijing, China). Sodium heptanesulfonate, phosphoric acid and acetic acid were of analytical grade(beijing, China), methanol was of chromatographic grade and doubly distilled water was prepared with Milli Q(Millipore, France). Adrenaline hydrochloride injection(stand 1 ml:1 mg, Batch Nos , and ) and noradrenaline bitartrate injection(stand 1 ml:2 mg, Batch Nos. 4A11002, and ) were purchased from a drugstore. 2.2 Apparatus During the experiment, three high performance chromatographs of liquid were used, which were a Shimadzu LC2010A, a Waters and an Agilent1100. The chromatographic columns used were Agilent Zorbax SB-C 18 (4.6 mm 250 mm, 5 μm PN: , SN: USCL010716), Yilite(E Batch No.5\120\6491) with bulking agent of Hypersil ODS2(4.6 mm 150 mm, 5 μm), Shiseido Finechemicals capcell C 18 (4.6 mm 250 mm, 5 μm Cat. No.90104), Yilite(E Batch No.5\120\6491) with bulking agent Hypersil ODS2 (4.6 mm 150 mm, 5 μm) and Inertsil ODS-3(4.6 mm 250 mm, 5 μm C/N: , S/N: 5FI86062). The other apparatus used were, a 1/100,000 electronic balance, a Mettler AE163 and a Mettler MP230 ph meter. 2.3 Preparation of Solutions Standard Solution of Reference Standard solution of adrenaline reference substance was prepared by dissolving 3.25 mg of adrenaline reference substance, accurately weighed, into a 25 ml volumetric flask with an acetic acid solution(1 25), and diluted quantitatively to obtain a solution containing adrenaline 0.1 mg/ml. Standard solution of noradrenaline bitartrate reference substance was prepared by dissolving 6.08 mg of noradrenaline bitartrate reference substance, accurately weighed, into a 25 ml volumetric flask with an acetic acid solution(1 25), and diluted quantitatively to obtain a solution containing adrenaline 0.2 mg/ml Standard Solution of Substitute for Reference Standard solution of the substitute for reference substance phenylephrine hydrochloride was prepared by dissolving 5.29 mg phenylephrine hydrochloride, accurately weighed, into a 25 ml volumetric flask with an acetic acid solution(1 25), and diluted quantitatively to obtain a solution containing phenylephrine hydrochloride about 0.2 mg/ml. 2.4 Quantitative Determination Chromatographic System and Conditions As the method in Ref. [3], a C 18 column packed with octadecyl silane bonded silica gel was used. The mobile phase of 0.14% sodium heptanesulfonate solution-methanol(65:35, volume ratio) was adjusted to make the ph value be 3.0±0.1 with phosphoric acid, and delivered at a flow rate of 1.0 ml/min. Column temperature was set at 35 ºC and 10 μl of samples was injected into the HPLC system. The UV wavelength was set at 282 nm for Adrenaline and 278 nm for phenylephrine hydrochloride. Theoretical plate number should be no less than 5000 when it is calculated according to adrenaline. As the method in Ref. [4], a C 18 column packed with octadecyl silane bonded silica gel was used. The ph value of mobile phase of 0.14% sodium heptanesulfonate solution-methanol(65:35, volume ratio) was adjusted to 3.0±0.1 with phosphoric acid, and delivered at a flow rate of 1.0 ml/min. Column temperature was set at 35 ºC and 10 μl of samples was injected into the HPLC system. The UV wavelength was

3 No.4 XIE Yuan-chao et al. 435 set at 278 nm for noradrenaline bitartrate and 274 nm for phenylephrine hydrochloride. Theoretical plate number should be no less than 5000 when it is calculated according to Adrenaline Quantitative Determination of Adrenaline Hydrochloride Injection with Substitute for Reference An appropriate amount(approximately adrenaline hydrochloride 3 mg) of adrenaline hydrochloride injection was weighed accurately, which was accurately placed in a 25 ml volumetric flask and dissolved and diluted with acetic acid solution(1 25). The solution was then mixed well, used as the assay sample solution. Under the conditions described in Section 2.4.1, 10 μl of the assayed sample solution was injected into the HPLC system for analysis. The chromatogram was detected and recorded at 282 nm. Moreover, the appropriate substitute for reference substance phenylephrine hydrochloride was weighted accurately and dissolved in an acetic acid solution(1 25) and then diluted quantitatively to obtain the substitute for reference substance solution containing phenylephrine hydrochloride 0.2 mg/ml. Then 10 μl of the substitute for reference substance solution was injected into the HPLC system and its chromatogram was detected and recorded at 278 nm. The content of adrenaline hydrochloride injection was calculated with the result from the external reference method being multiplied by a correction factor Quantitative Determination of Noradrenaline Bitartrate Injection with Substitute for Reference An appropriate amount(approximately noradrenaline bitartrate 4 mg) of noradrenaline bitartrate injection was weighed accurately and was accurately placed in a 25 ml volumetric flask and dissolved and diluted with an acetic acid solution(1 25). The solution was then mixed well, used as the assayed sample solution. Under the condition described in Section 2.4.1, 10 μl of the assayed sample solution was injected into the HPLC system for analysis. The chromatogram was detected and recorded at 278 nm. Moreover, the appropriate substitute for reference substance phenylephrine hydrochloride was weighted accurately and dissolved with an acetic acid solution(1 25) and then diluted quantitatively to obtain the substitute for reference substitute solution containing phenylephrine hydrochloride 0.2 mg/ml. Then 10 μl of the substitute for reference substitute solution was injected into the HPLC system and its chromatogram was detected and recorded at 274 nm. The content of noradrenaline bitartrate injection was calculated with the result from the external reference method being multiplied by a correction factor of The HPLC of substitute for reference substance, reference substance, adrenaline injection and noradrenaline bitartrate injection are shown in Fig.1. Fig.1 HPLC of negative sample(a), substitute reference substance(b), adrenaline reference substance(c), noradrenaline bitartrate reference substance(d), adrenaline hydrochloride injection(e) and noradrenaline bitartrate injection(f) 2.5 Validation of the Method Investigation of the Linearity The standard solutions(section 2.3) of a series of amounts of the adrenaline reference substance, phenylephrine hydrochloride were, respectively, injected into the HPLC under the chromatographic conditions described in Section The calibration curve of adrenaline was linear in a range of μg and its representative linear equation(n=5) relating y(peak area) to x(amount μg) was y= x (R=1.0000). The calibration curve for phenylephrine hydrochloride was linearly changed in a range of μg and its representative linear equation

4 436 CHEM. RES. CHINESE UNIVERSITIES Vol.25 (n=5) was y= x (r=0.9998). The standard solutions(section 2.3) of a series of amounts of the noradrenaline bitartrateand reference substance, phenylephrine hydrochloride, was injected into the HPLC under Section decribed chromatographic conditions. The calibration curve for noradrenaline bitartrate was linear in a range of μg and its representative linear equation(n=5) was y= x (R=1.0000). The calibration curve for phenylephrine hydrochloride was linear in a range of μg and its representative linear equation (n=5) was y= x (r=1.0000) Determination of f Value Typically, 20, 15, 10, 6 and 2 μl of the standard solution phenylephrine hydrochloride(section 2.3.2) were, respectively, injected into the HPLC under Section described chromatographic conditions. We used the amount as X-axis and the peak area as Y-axis. The calibration curves of the reference substance and its substitute were then determined. After the intercepts of the calibration curves were adjusted to 0, the correction factor( f ) was calculated with the ratio of the two rates of the standard curves(k) [2]. The correction factor( f ) of Adrenaline was Generally, 20, 15, 10, 6 and 2 μl of the standard solution of phenylephrine hydrochloride(section 2.3.2) were, respectively, injected into the HPLC under Section described chromatographic conditions. Likewise the correction factor( f ) of Noradrenaline bitartrate was Precision and Accuracy of the Method The precision of the proposed method(section 2.4.1) was determined by selecting the standard solutions of adrenaline reference substance(0.1 mg/ml) and phenylephrine hydrochloride(0.2 mg/ml) that were injected 6 times repeatedly to record the peak areas of adrenaline and phenylephrine hydrochloride. The RSD value of precision was 0.4% for adrenaline and 0.6% for phenylephrine hydrochloride, respectively. The precision of the proposed method(section 2.4.1) was determined by selecting the standard solutions of noradrenaline bitartrate reference substance(0.2 mg/ml) and its substitute phenylephrine hydrochloride(0.2 mg/ml) that were injected 6 times repeatedly to record the peak areas of noradrenaline bitartrate and phenylephrine hydrochloride. The RSD value of precision was 1.0% for noradrenaline bitartrate and 0.6% for phenylephrine hydrochloride. The accuracy was evaluated by parallel sextuple determinations of adrenaline in the same batch number adrenaline hydrochloride injection with phenylephrine hydrochloride according the assay method as described in Section The content of the sample was 90.8% and the RSD value of accuracy was 1.0%. The accuracy was evaluated by parallel sextuple determinations of noradrenaline bitartrate injection in the same batch number 4A11002 noradrenaline bitartrate injection phenylephrine hydrochloride according the assay method as described in Section The content of the sample was 93.2% and the RSD value of accuracy was 1.1% Recovery Experiment The adrenaline solutions with concentrations of 1.0, 1.3 and 1.6 mg/ml were prepared in triplicate each by weighing sufficiently accurate quantity of adrenaline reference substance, which were subsequently dissolved and diluted quantitatively within acetic acid solution(1 25). The solution of phenylephrine hydrochloride with a concentration of 2.0 mg/ml was prepared in duplicate by accurately weighing quantity of phenylephrine hydrochloride, which was then dissolved and quantitatively diluted with acetic acid solution(1 25). Using phenylephrine hydrochloride for assaying the content of adrenaline with the method described in Section 2.4.2, the recoveries were calculated(table 1). Table 1 Recovery of adrenaline with the aid of phenylephrine hydrochloride and correction factor f Added Detected Recovery(%) Average recovery(%) RSD(%) The noradrenaline bitartrate with solutions concentrations of 2.0, 2.4 and 2.9 mg/ml were prepared in triplicate each by weighing accurately sufficient quantity of noradrenaline bitartrate reference substance, which were subsequently dissolved and diluted quantitatively with an acetic acid solution(1 25). The

5 No.4 XIE Yuan-chao et al. 437 solution of phenylephrine hydrochloride with concentration of 2.0 mg/ml were prepared in duplicate by accurately weighing quantity of phenylephrine hydrochloride, which was then dissolved and quantitatively diluted with an acetic acid solution(1 25). Using the substitute for reference substance phenylephrine hydrochloride for assaying with the method described in Section 2.4.3, the recoveries were calculated(table 2). Table 2 Recovery of noradrenaline bitartrate with the aid of phenylephrine hydrochloride and correction factor f Added Detected Recovery(%) Average recovery(%) RSD(%) Inspection of the Stability of the Solutions The stabilities of the assayed sample solution and the standard solution of the substitute for reference substance were monitored by measuring the peak areas. The assayed sample solution of adrenaline(batch No ) and noradrenaline bitartrate injection(batch No.4A11002) singularly was determined at 1st, 2nd, 4th, 6th and 8th hour, respectively. The result indicates that the assayed sample solutions of adrenaline and noradrenaline bitartrate injections were stable and their RSD values were 0.2% and 0.2%, respectively, in 8 h. The solution of the substitute for reference substance was determined on 1st, 2nd, 4th, 6th and 8th hour, respectively. The result indicate that the solution of phenylephrine hydrochloride was stable and its RSD value were 0.7% in 8 h Limit of Detection and Limit of Quantitation The limit of detection(s/n 3:1) and the limit of quantitation(s/n 10:1) for adrenaline were 2.6 and 9.2 μg/ml, respectively. The limit of detection(s/n 3:1) and the limit of quantitation(s/n 10:1) for noradrenaline bitartrate were 4.9 and 17 μg/ml, respectively. The limit of detection(s/n 3:1) and the limit of quantitation(s/n 10:1) for phenylephrine hydrochloride were 6.4 and 19 μg/ml, respectively. 3 Results and Discussion 3.1 Selection of Suitable Working Wavelength The UV spectra of adrenaline, noradrenaline bitartrate and phenylephrine hydrochloride are illustrated in Fig.2. The maximum absorbance of adrenaline was at about 282 nm and that of phenylephrine hydrochloride was at about 278 nm. The maximum absorbance of noradrenaline bitartrate was at about 280 nm. Therefore the detecting wavelength was chosen at their nearby maximum absorbance so as to reduce the error of determination. Fig.2 UV spectra of adrenaline, noradrenaline bitartrate and phenylephrine hydrochloride S 1 : adrenaline; S 2 : noradrenaline bitartrate; T: phenylephrine hydrochloride. 3.2 Analysis of Real Samples Analysis of Real Samples of Adrenaline Hydrochloride Injection with the Substitute for Reference The contents of 3 batches of adrenaline hydrochloride injection(batch Nos , and ) determined with the reference substance and the substitute for reference substance under Section are shown in Table 3. According to the t-test, we found that there were no significant differences (P>0.05) between the contents determined by the two methods. Table 3 Adrenaline hydrochloride injection content Batch Content by reference substance(%) Content by substitute for reference substance(%) Analysis of Real Samples of Noradrenaline Bitartrate Injection with the Substitute for Reference The contents of 3 batches of noradrenaline bitartrate injection(batch Nos , and 4A11002) determined with the reference substance

6 438 CHEM. RES. CHINESE UNIVERSITIES Vol.25 and the substitute for reference substance under Section are shown in Table 4. According to the t-test, we found that there were no significant differences (P>0.05) between the contents determined by the two methods. Table 4 Noradrenaline bitartrate injection content Batch Content by reference substance(%) Content by substitute for reference substance(%) The correction factor f was introduced into the determination of adrenaline and noradrenaline bitartrate with the substitute for reference substance. The results of the content of adrenaline injection and noradrenaline bitartrate injection determined with the re- ference substance and the substitute for reference substance were approximate to each other. Therefore the method developed in the present study is precise, accurate and feasible for the quality control of adrenaline injection and noradrenaline bitartrate injection in market. References [1] European Pharmacopoeia Commission, European Pharmacopeia 5.0, 2004, 1651 [2] Liu M., Hu C. Q., Chin. Pharm. Anal., 2004, 24, 380 [3] National Pharmacopeial Commission, The Chinese Pharmacopeia Second Portion, China, 2005, 334 [4] National Pharmacopeial Commission, The Chinese Pharmacopeia Second Portion, China, 2005, 418