First Derivative Spectrophotometric Determination of Hostacycline and Oxytetracycline – Applications
A. Raghavendra Guru Prasad1* and V. Suryanarayana Rao2
1Department of Chemistry, ICFAI University, Dehradun, U.K., India
2Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India.
*Corresponding Author E-mail: guruprasadar@yahoo.co.in
ABSTRACT:
Simple, selective and sensitive first order spectrophotometric method has been developed for the determination of hostacycline and oxytetracycline at pH 3.5. Linear calibration plots are obtained in the range of 10 to 60µg/ml for each of the tetracycline. The method is also extended for the resolution of their binary mixtures by using the zero-crossing method for evaluation of the derivative signal. The corresponding calibration plot is linear in the rage 10 µg/ml to 25µg/ml for each of the tetracycline. Regression equation, correlation coefficient and percentage recovery is reported in each case. The developed method has been successfully applied for the determination of hostacycline and oxytetracycline in pharmaceutical formulations, urine and honey. A notable advantage of the proposed method is that the method can be applied without any pretreatment of the samples.
KEYWORDS: Hostacycline; Oxytetracycline; Spectrophotometry; Zero crossing method; Resolution.
INTRODUCTION:
The tetracycline antibiotics are widely used in the treatment of infections of respiratory and urinary tracts. So also recent reports on antibiotics suggest that they are widely used in modern agriculture. They are frequently used in veterinary medicine, animal nutrition and as feed additives.1
The property of tetracyclines to form complexes with metal ions is extensively utilized for the analytical determination of tetracyclines.2 A number of such methods for the determination of tetracyclines are reported from these laboratories.3-5 In these methods tetracyclines show identical analytical behavior. Hence a resolution of binary mixtures of tetracyclines cannot be achieved by employing these procedures.
In the recent years there has been a growing interest in the analytical applications of derivative spectrophotometry. A number such methods are reported for the analytical determination of pharmaceutical formulations.6-10 A brief review of literature suggests that only few methods are reported for the simultaneous determination of tetracyclines11-14.
These methods are very expensive and include complicated procedure. Even the official monographs of the United States Pharmacopoeia15 describe methods for the determination of tetracyclines in pharmaceutical formulations using high-performance liquid chromatography (HPLC), but each compound requires different conditions for determination. The biological assay proposed by the British Pharmacopoeia16 is time-consuming and not suitable for routine analysis.
Antibiotic residues on foods 17-20 are of great concern because of the possible toxic or allergic reactions. In response to these concerns, health authorities insist that no tetracycline residues should be present in foods. Honey can become contaminated with tetracyclines21, 22 due to the previous treatment of the beehive.. Hence honey requires tetracycline free certification.23 So monitoring tetracycline in foods, honey and biological samples such as urine is an important aspect.. In this article, the authors present first derivative spectrophotometric methods for the determination of hostacycline (HTC) and oxytetracycline (OTC). The first derivative spectra are also used to resolve the binary mixtures containing HTC and OTC. The applicability of the method is successfully tested for the determination of HTC and OTC in urine, honey and in pharmaceutical samples.
EXPERIMENTAL:
Spectral measurements are performed with an Elico SL 164 UV-Visible spectrophotometer. The pH measurements are made using an Elico pH meter. HTC and OTC each of concentration 0.5mg/ml are prepared in double distilled water.
General procedure for the determination of HTC/OTC
5 ml of the buffer solution of required pH and aliquots of HTC/OTC solutions of concentration 0.5mg/ml are taken in a 10ml volumetric flask. The solution is made up to the mark with double distilled water. The spectrum of the reaction mixture is recorded against the blank solution containing no drug.
Resolution of mixtures of HTC/OTC
A series of samples were prepared in 10ml volumetric flasks to contain different aliquots of HTC and OTC each of concentration 0.5mg/ml. 5ml of buffer solution of required pH is taken into the flask and the solution is diluted up to the mark with double distilled water. The spectrum of the reaction mixture is recorded against a blank solution containing no drug.
RESULTS AND DISCUSSION:
Effect of pH
pH of the solution is varied from 1 to 8. It is observed that HTC and OTC give similar zero order absorption spectra. Hence the author employed first derivative spectrophotometry to determine one drug in the presence of the other. The corresponding first order amplitudes are recorded. The variation of first order amplitudes with pH is shown in the Fig. 1 and Fig 2. A close examination of the above figures reveals only small changes in amplitudes in the pH range 3 to 4. So a pH of 3.5 is selected for further studies.
Fig 1 -Effect of pH on first derivative amplitude
A) At 282.5 nm; B) At 380.5 nm
Fig 2-Effect of pH on first derivative amplitude
A) At 281.5 nm; B) At 377.0 nm
Determination of HTC/OTC:
The first order spectra of HTC and OTC at different concentrations are shown in the Fig. 3 and Fig. 4 respectively. The HTC and OTC concentrations can be determined by measuring the first order signals at 282.5, 312.5, 331, 346.5, 380.5 and 281.5, 310, 344, 377nm respectively. The linear plot (Fig. 5 and Fig 6) obtained between the first order signal and concentration suggests that the method can be successfully used for the determination of HTC/OTC in the concentration range 10 to 60 μg/ml. The corresponding regression parameters at various wavelengths are shown in the Table 1 and Table 2.
Fig. 3 First derivative spectra at various concentrations of hostacycline
A. 10 mg/ml; B. 20 mg/ml; C. 30 mg/ml; D. 40 mg/ml; E. 50 mg/ml; F. 60 mg/ml
Fig.4 First derivative spectra at various concentrations of oxytetracycline
A. 10 mg/ml; B. 20 mg/ml; C. 30 mg/ml; D. 40 mg/ml; E. 50 mg/ml; F. 60 mg/ml
Fig. 5 Calibration plots for hostacycline at different wavelengths
Primary Y-axis: A) 282.5 nm; C) 331.0 nm; E) 380.5 nm;
Secondary Y-axis: B) 312.5 nm; D) 346.5 nm.
Fig. 6 Calibration plots of oxytetracycline at different wavelengths
Primary Y-axis: A) 281.5 nm; B) 310 nm;
Secondary Y-axis: C) 344 nm; D) 377 nm.
|
Regression equation |
Correlation coefficient |
|
1D282.5 = 0.0042C + 0.0109 |
0.9985 |
|
1D312.5 = -0.0006C+0.0013 |
0.9987 |
|
1D331.0 = -0.0012C + 0.0109 |
0.9973 |
|
1D346.5 = -0.0013C + 0.0009 |
0.9938 |
|
1D380.5 = 0.0045C + 0.0107 |
0.9999 |
TABLE 2- Regression parameters for determination of oxytetracycline; C = [OTC] in µg/ml
|
Regression equation |
Correlation coefficient |
|
1D281.5 = 0.0037C + 0.0041 |
0.9989 |
|
1D310.0 = -0.0004C + 0.0012 |
0.9979 |
|
1D344.0 = -0.0011C + 0.0013 |
0.9992 |
|
1D377.0 = 0.0039C + 0.0093 |
0.9991 |
Resolution of HTC and OTC in their binary mixtures:
Resolution of HTC and OTC cannot be performed using their zero order spectra. As revealed by the first order spectra (Fig. 7), the zero crossing wavelengths for HTC and OTC are 358 and 351nm respectively. The simultaneous determination of HTC and OTC is performed by zero crossing wavelength method. Hence the concentrations of HTC and OTC can be determined measuring first order amplitudes at 351 and 358 nm respectively. (Fig. 8) The calibration plot drawn between first order amplitude and concentration of HTC/OTC is shown in the Fig.9. The corresponding regression parameters are shown in the Table. 3.
APPLICATIONS:
1. Pharmaceutical formulations:
The above methods are applied for the determination of HTC and OTC in pharmaceutical formulations containing HTC and OTC and in their synthetic mixtures. The percentage recoveries are evaluated in each case and are shown in the Tables 4, 5 and 6. The results are found to satisfactory.
Fig. 7 (A) First derivative spectra of hostacycline (25 mg/ml)
(B) First derivative spectra of oxytetracycline (25 mg/ml)
(C)First derivative spectra of binary mixture of hostacycline (25mg/ml) and oxytetracycline (25 mg/ml) Zero cross points: 1 - hostacycline (358 nm) and 2 - oxytetracylcine (351 nm)
Fig. 8 First derivative spectra of binary mixture of hostacycline and oxytetracycline at different concentrations.
A) 10 mg/ml; B) 15 mg/ml; C) 20 mg/ml; D) 22.5 mg/ml; E) 25 mg/ml of each.
Fig. 9 Effect of HTC/OTC concentration on first derivative amplitude in their mixtures
A) Calibration plot for OTC at 358.0 nm
B) Calibration plot for HTC at 351.0 nm
TABLE 3-Regression parameters for determination of HTC or OTC; C = [HTC or OTC] in µg/ml
|
Tetracycline
|
Regression equation |
Correlation coefficient |
|
OTC |
1D358.0 = 0.002C – 0.0047 |
0.9973 |
|
HTC |
1D351.0 = -0.0011C - 0.0023 |
0.9992 |
|
Signal measured |
Sample |
Labelled amount mg/tab or capsule |
Amount found mg/tab or cap |
Recovery (%) |
|
1D282.5 |
Linemettea |
500 |
495 |
99 |
|
Resticlinb |
250 |
248 |
99.2 |
|
|
1D312.5 |
Linemettea |
500 |
504 |
100.8 |
|
Resticlinb |
250 |
247 |
98.8 |
|
|
1D331.0 |
Linemettea |
500 |
496 |
99.2 |
|
Resticlinb |
250 |
246 |
98.4 |
|
|
1D346.5 |
Linemettea |
500 |
506 |
101.2 |
|
Resticlinb |
250 |
254 |
101.6 |
|
|
1D380.5 |
Linemettea |
500 |
505 |
101.0 |
|
Resticlinb |
250 |
498 |
99.6 |
2. Urine:
The human body eliminates 30 to 60% of initially administered unchanged tetracyclines through urine during the first 24 hours. The author directly applied the above methods for the determination of HTC and OTC in urine without any pretreatment. In order to eliminate the background of urine a dilution of 1:30 is performed. The results are presented in the Table. 7.
|
Signal measured |
Sample |
Labelled amount mg/tab or capsule |
Amount found mg/tab or capsule |
Recovery (%) |
|
1D344.0 |
Oxytetrcyclinea |
100 |
102.5 |
102 |
|
Terramycinb |
100 |
98 |
98 |
|
|
1D281.5,377.0 |
Oxytetrcyclinea |
100 |
98 |
98 |
|
Terramycinb |
100 |
101 |
101 |
|
|
1D310.0 |
Oxytetrcyclinea |
100 |
104 |
104 |
|
Terramycinb |
100 |
101 |
101 |
a = Sarabhai Chemicals Ltd., Vadodara, India; b = Pfizer India Ltd., Mumbai, India.
OTC to HTC ratio |
OTC |
HTC |
||||
|
Taken µg/ml |
Found µg/ml |
Recovery % |
Taken µg/ml |
Found µg/ml |
Recovery % |
|
|
2:2 |
10 |
10.2 |
102 |
10.0 |
10.4 |
104 |
|
2:4.5 |
10 |
10.3 |
103 |
22.5 |
22.2 |
98 |
|
4.5:2 |
22.5 |
22.8 |
101 |
10 |
9.7 |
97 |
|
4:5 |
20 |
18.9 |
94 |
25 |
26.1 |
104 |
|
5:2 |
25 |
24.6 |
98.4 |
10 |
9.8 |
98 |
TABLE 7-Determination of hostacycline in urine
|
Sample |
Signal measured |
Added (µg/ml) |
Found (µg/ml) |
Recovery (%) |
|
Urine (1+30 dilution) + hostacycline |
1D380.5 |
15 |
15.3 |
102 |
Determination of oxytetracycline in urine
|
Sample |
Signal measured |
Added (µg/ml) |
Found (µg/ml) |
Recovery (%) |
|
Urine (1+30 dilution) + oxytetracycline |
1D377.0 |
12 |
12.5 |
104 |
3. Honey
The proposed method can be employed to determine the amounts of HTC and OTC in honey without any pretreatment. A dilution of 1:20 is recomended to eliminate the background of honey. The results are presented in the Table. 8
TABLE 8- Determination of hostacycline in honey
|
Sample |
Signal measured |
Added (µg/ml) |
Found (µg/ml) |
Recovery (%) |
|
Honey (1+20 dilution) + hostacycline |
1D380.5 |
15 |
15.3 |
102 |
Determination of oxytetracycline in honey
|
Sample |
Signal measured |
Added (µg/ml) |
Found (µg/ml) |
Recovery (%) |
|
Honey (1+20 dilution) + oxytetracycline |
1D377.0 |
12 |
12.4 |
103 |
CONCLUSIONS:
The method presented by authors permits simple, sensitive and accurate first derivative spectrophotometric determination of HTC and OTC and their resolution in synthetic binary mixtures. Satisfactory results are obtained when these methods are applied to the pharmaceutical formulations containing these drugs. An advantage of the method lies in the fact that these procedures may be directly employed to urine and honey without any pretreatment.
REFERENCES:
1. W. How and E. Wang. Liquid chromatographic determination of tetracycline antibiotics at an electrochemically pre-treated glassy carbon electrode, Analyst. 1989; 114; 699-702.
2. Roushdi IM, Ibrahim el-SA, Beltagy YA, and Issa A. Colometric methods for the estimation of tetracycline hydrochloride and oxytetracycline hydrochloride, Die Pharmazie 1973; 28(4); 236-7.
3. Siva Chandra, Y., Suryanarayana Rao, V. Rama Murthy. P. S., Determination of hostacycline and doxycycline using Thorium( IV) as spectrophotometric reagent. Indian J. Pharm. Sci. 1996; 157-159.
4. Suryanarayana Rao, V and Rama Devi, B. Spectrophotometric determination tetracycline hydrochloride, Indian Drugs. 1993; 30; 531-533.
5. MB Rao, PS Ramamurthy, VS Rao, Basanti Rao M., Suryanarayana Rao. V. (1996), Spectrophotometic determination of oxytetracycline in pharmaceutical dosage forms. 1996; Volume : 58; Issue : 6; 254-255
6. Abdine, H.H., Gazy, A.A., Abdel-Hay, M.H. Simultaneous determination of melatonin-pyridoxine combination in tablets by zero-crossing derivative spectrophotometry and spectrofluorimetry. J Pharm Biomed Anal. 1998; 17(3); 379-86.
7. Nevado, B.J.J., Flores, R.J., Llerena, V.M.J., Fresenius, J. Simultaneous determination of tartrazine, riboflavine, curcumin and erythrosine by derivative spectrophotometry. Fresenius Journal of Analytical Chemistry. 1994; 350(10-11); 610-616.
8.
J. A. Murillo, J. M.
Lemus and L. F. García. Simultaneous determination of the binary mixtures of
cefsulodin and clavulanic acid by using first-derivative spectrophotometry. Journal of Pharmaceutical and
Biomedical Analysis
Volume 13; Issue 6;
1995; 769-776.
9. V. Rodenas, M. S García, C. Sánchez-Pedreńo, M. I. Albero. Simultaneous determination of propacetamol and paracetamol by derivative spectrophotometry. Talanta. 2000; Volume 52; Issue 3; 517-523.
10. Morelli, B. Zero-crossing derivative spectrophotometric determination of mixtures of cephapirin sodium and cefuroxime sodium in pure form and in injections. Analyst. 1988; 113(7); 1077-1083.
11. Yan Xiong, Houjiang Zhou, Zhujun Zhang, Deyong He, Chao He. Molecularly imprinted on-line solid-phase extraction combined with flow-injection chemiluminescence for the determination of tetracycline, Analyst. 2006; 7; 829-834.
12. Yuanling Liang, Richard E. Simon, M. Bonner Denton. Utilization of a scientifically operated charge-coupled device detector for high-performance thin-layer chromatographic analysis of tetracyclines. Analyst. 1999; 11; 1577-1582.
13. Liawruangrath Saisunee, L. Boonsom, Surasak and Ruengsitagoon Wirat. Flow Injection Spectrophotometric Determination of Tetracycline in a Pharmaceutical Preparation by Complexation with Aluminium (III), Anal Sci. 2006; Vol.22; No.1; 15-19.
14. José L. Rufino; Patrícia L. Weinert; Helena R. Pezza; Leonardo Pezza. Flow-injection spectrophotometric determination of tetracycline and doxycycline in pharmaceutical formulations using chloramine-T as oxidizing agent, Quím. Nova. 2009; Vol.32; No.7 Săo Paulo.
15. United States Pharmacopeia, 26th ed., Rockvillle, 2003.
16. Britsh Pharmacopoeia, HM Stationery Office: London, 1988.
17. Ashworth RB. Liquid chromatographic assay of tetracyclines in tissues of food producing animals. J Assoc Off Anal Chem 1985; 68; 1013-1018.
18. E. E. Martinez and W. Shimoda, Liquie chromatographic determination of tetracycline residues in animal feeds. J. Assoc. Off. Chem. 1988; 71; 477-480.
19. Hiroyuki Nakazawa, Shigeru Ino, Kayoko Kato, Takaho Watanabe, Yuko Ito and Hisao Oka. Simultaneous determination of residual tetracyclines in foods by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry, Journal of Chromatography B: Biomedical Sciences and Applications, 1999; Volume 732; Issue 1; 55-64.
20. M.C. Carson. Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography particle beam mass spectrometry, Journal of Chromatography B. 1998; 712; 113-117.
21. Corner J, Gochnauer TA. The persistence of tetracycline activity in medicated syrup stored by wintering honeybee colonies, J Apic Res. 1971; 10; 67-71.
22. Seu-Ping Khong, Yves-Alexis Hammel, Philippe A. Guy. Analysis of tetracyclines in honey by high-performance liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry. 2005; Vol. 19; Issue 4; 493 – 502.
23. Sporns P, Kwan S, Roth LA. HPLC analysis of oxytetracycline residues in honey, J Food Prot. 1986; 49; 383-388.
Received on 22.07.2009 Modified on 28.02.2010
Accepted on 05.03.2010 © AJRC All right reserved
Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 220-224