INTRODUCTION:

7-ADCA (7-Amino deacetoxy cephalosporanic acid) is an important intermediate for preparing cephalosporin antibiotics, is prepared by a novel bioprocess in which a transformed Penicillium chrysogenum strain is cultured in the presence of an adipate feedstock to produce adipoyl-6-APA (6-amino penicillanic acid); and the in situ expression of an expandase gene, e.g., from Streptomyces clavuligerus, with which the P. chrysogenum has been transformed, converts the adipoly-6-APA by ring expansion to adipoyl-7-ADCA. The final product 7-ADCA, is then prepared by cleavage of the adipoyl side chain using an adipoyl acylase. The entire synthesis, accordingly, is carried out using bioprocesses, and is efficient and economical.

A very few physico-chemical methods appeared in the literature for the assay of 7-ADCA in biological fluids and pharmaceutical formulations.

The methods so far reported include HPLC^1-8, CE⁹, GC-MS^10-11, and UV-Visible spectrophotometric methods.^12,13

Existing analytical methods reveal that relatively little attention was paid in developing visible spectrophotometric methods by exploiting thoroughly the analytically useful functional groups in 7-ADCA. Hence there is a need to develop sensitive and flexible visible spectrophotometric methods, which prompted the author to choose 7-ADCA for the investigation. Based on the different chemical reactions two methods have been developed. These methods were based on the reactivity of 7-ADCA with reagents such as NBS/PMAP/SAc(M_a); Hae/CAT (M_b); NBS/CB (M_c). All these methods have been extended to pharmaceutical formulations as well. The author has developed two simple and sensitive UV methods (CH₃OH as solvent) and adopted it as a reference method to compare the results obtained by proposed methods. The analytical utility of the proposed chromogenic reagents

EXPERIMENTAL:

Instruments used: An Elico, UV – Visible digital spectrophotometer with 1cm matched quartz cells were used for the spectral and absorbance measurements. An Elico LI-120 digital pH meter was used for pH measurements.

Preparation of standard drug solutions: A 1 mg/ml solution was prepared by dissolving 100 mg of pure 7-ADCA in 100ml of distilled water and this stock solution was diluted step wise with distilled water to get the working standard solutions of concentration.

Preparation of reagents: All the chemicals and reagents used are of analytical grade and solutions were prepared in triply distilled water

Method M_a: NBS solution (Loba; 0.088%, 4.94x10^-3 M): Prepared by dissolving 88 mg of N-Bromo succinimide in 100 ml of distilled water and standardised iodometrically.

PMAP solution (Wilson Labs; 0.3%, 8.71x10^-3M): Prepared by dissolving 300mg of p-N-methyl aminophenol sulphate in 100 ml of distilled water.

SAc solution: Prepared by dissolving 1gm of p-sulphanilic acid in 100ml of 0.2M HCl.

Acetic acid (Qualigens, 5% v/v, 8.75x10^-1 M): 5 ml of glacial acetic acid was diluted to 100 ml with distilled water.

Method M_b: Haematoxylin solution (Aldrich, 0.2%, 6.605x10^-3M): Prepared by dissolving 200 mg of haematoxylin in 100ml of methanol.

CAT solution (BDH, 0.4%, 1.42x10^-2M): Prepared by dissolving 400mgs of Chloramine-T (CAT) in 100ml of distilled water and standardized.

Buffer solution pH7.0: Prepared by mixing 390ml of 0.067M potassium hydrogen phosphate (BDH) and 610ml of 0.067M disodium hydrogen phosphate (BDH) and the pH of the solutions was adjusted to 7.0

Method M_c: NBS solution (Loba; 0.01%, 5.618x10^-4 M): Prepared by dissolving 50 mg of N-Bromo succinimide in 500 ml of distilled water and standardized iodometrically.

CB solution (Chroma; 0.005%, 5.497x10^-4M): Prepared by dissolving 50mg of celestine blue in 1000 ml of distilled water Hydrochloric acid (E.Merck, 5M): Prepared by diluting 217.5 ml of concentrated HCI to 500 ml with distilled water and standardized

Recommended Procedures:

Method M_a: Aliquots of standard 7-ADCA solution (1.0-5.0 ml, 200 mg/ml) were transferred into a series of 25ml-calibrated tubes. Then 0.5ml (8.75x10^-1M) of AcOH and 2 ml (4.94x10^-3M) of NBS solutions were added and kept aside for 15 min. at room temperature. Then 1.5 ml (8.71x10^-3M) of PMAP solution was added. After 2 min 2.0 ml (1.16x10^-2M) of SAc solution was added. The volume was made up to the mark with distilled water. The absorbance was measured after 10 min. at 560 nm against distilled water. A blank experiment was also carried out omitting the drug. The decrease in the absorbance and in turn the drug concentration was obtained by subtracting the absorbance of the test solution from the blank. The amount of 7-ADCA was computed from its calibration graph.

Method M_b: To a series of 25ml-graduated test tubes, 1ml each of Haematoxylin and Chloramine-T, 15 ml of buffer (pH7.0) solutions were added successively. The mixture was set aside for 20 min. Then added aliquots of drug with in Beer’s law limits (0.5-3.0 ml, 100 mg/ml) and kept in a water bath at 70°C for 20 min. The test tubes were removed from the water bath, cooled to the room temperature. The contents in tube were diluted to 25 ml with distilled water and the absorbance read at 630 nm within the stability period (immediate 40min). The amount of drug was deduced from its standard calibration curve graph.

Method M_c: Aliquots of standard 7-ADCA solution (0.5-3.0 ml, 20 mg/ml) were transferred into a series of 25 ml calibrated tubes. Then 1.25 ml (5.0M) of HCI and 2.5 ml (5.618x10^-4 M) of NBS were added. The volume was brought to15ml with distilled water. After 10min, 10ml (5.50x10^-4M) of CB solution was added and mixed thoroughly. The absorbance was measured after 5 min at 520 nm against distilled water. The blank (omitting drug) and dye (omitting drug and oxidant) solutions were prepared in a similar manner and their absorbance was measured against distilled water. The decrease in absorbance corresponding to consumed NBS and in turn the drug concentration was obtained by subtracting the decrease in absorbance of the test solution (dye-test) from that of the blank solution (dye-blank). The amount of ADCA was computed from its calibration graph

Reference Method¹³: An accurately weighed portion of the powdered tablets equivalent to 100 mg of drug was dissolved in 30 ml of isopropyl alcohol, shaken well and filtered and the filtrate was diluted to 100 ml with isopropyl alcohol to get 1mg/ml solution of drug in formulations. Five ml of this solution was further diluted to 200 ml to get 25 mg/ml solution. The absorbance of the solution was determined at l_max 229 nm. The quantity of the drug was computed from the Beer’s law plot of the standard drug in isopropyl alcohol.

For pharmaceutical formulations: An accurately weighed portion of tablet content equivalent to about 100 mg of 7-ADCA was transferred into a 100 ml volumetric flask. Added about 80 ml of warm isopropyl alcohol and shaken well for about 20 min. The contents were diluted with isopropyl alcohol up to the mark and mixed thoroughly. The solution was filtered. The filtrate was evaporated to dryness. The residue was used for the preparation of formulation solutions for different methods as given under standard solutions preparations. These solutions were analyzed as under procedures described fro bulk solutions.

RESULTS AND DISCUSSIONS:

Spectral Characteristics: In order to ascertain the optimum wavelength of maximum absorption (l_max) of the colored species formed in the above methods, specified amounts of 7-ADCA were taken and colors were developed separately by following the above procedures. The amounts of 7-ADCA present in total volume of colored solutions were 8 mg/ml (M_a), 4 mg/ml (M_b), 0.8 mg/ml (M_c). The absorption spectra were scanned on a spectrophotometer in the wavelength region of 340 to 900 nm against similar reagent blank or distilled water. The reagent blank absorption spectrum of each method was also recorded against distilled water. The absorption curves of the colored species in each method show characteristics absorption maxim where as the blank in each method has low or no absorption in this region.

Optimum conditions fixation in procedures: The optimum conditions for the color development of methods (M_a, M_b, and M_c)were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of the colored species. The following experiments were conducted for this purpose and the conditions so obtained were incorporated in recommended procedures.

Method M_a(NBS/PMAP/SAc): This is an indirect spectrophotometric method, which involves two steps, oxidation of the ADCA with NBS (first step) and estimation of the unconsumed NBS with PMAP- SAc reagent (second step).

In the first step, the volume of NBS required for oxidation of drug, the time and temperature for oxidation of the drug and volume of acetic acid were established through control experiments. In the second step, the volume of PMAP and the intermittent time between additions, volume of SAc and the solvent for final dilution were found by varying one parameter at a time and the optimum conditions are incorporated in Table 1a.

Method M_b(Haematoxylin/CAT): The optimum conditions in this method were found basing on the study of the effects of various parameters such as volume of 6.60x10^-3 M haematoxylin solution and 1.42x10^-2M CAT solution, pH of the buffer to achieve maximum absorbance, heating time and temperature for maximum color development, the stability and intensity of the color species after final dilution were established by measuring absorbance at 580 mm and recorded in the Table 1b.

Method M_c(NBS/CB): The procedure involves two steps. The first step in the procedure is the reaction of 7-ADCA with an excess of NBS giving products involving oxidation, substitution or addition and the estimation of unreacted NBS using a known excess of CB (second step). The excess dye remaining was then measured with a spectrophotometer. The effect of reagent concentration (acidity, NBS and CB), waiting period in each step with respect to maximum sensitivity, minimum blank, adherence to Beer’s law, reproducibility and stability of final color were studied by means of control experiments varying one parameter at a time and the optimum conditions are incorporated in Table 1c.

Optical Characteristics: In order to test whether the colored species formed in the above methods, adhere to Beer’s law the absorbance’s at appropriate wave lengths of a set of solutions containing varying amounts of 7-ADCA and specified amounts of reagents (as given in the recommended procedures for each method) were recorded against the corresponding reagent blanks. The Beer’s law plots of these systems are recorded against the corresponding reagent blanks. The Beer’s law plots of these systems are recorded graphically. Beer’s law limits, molar absorptivity, Sandell’s sensitivity and optimum photometric range (Table 2) for 7-ADCA in each method developed. With mentioned reagents were calculated. Least square regression analysis was carried out for getting the slope, intercept and correlation coefficient values.

Precision: The precision of each proposal methods was ascertained from the absorbance values obtained by actual determination of six replicates of a fixed amount of 7-ADCA in total solution. The percent relative standard deviation and percent range of error (at 0.05 and 0.01 confidence limits) were calculated for the proposed methods (Table 2).

Accuracy: To determine the accuracy of each proposed method, different amounts of bulk samples of 7-ADCA within the Beer’s law limits were taken any analyzed by the proposed method. The results (% error) are recorded in Table 2.

Interference studies: The effect of wide range of excipients and other active ingredients usually present in the formulations for the assay of 7-ADCA in methodsunder optimum conditions were investigated. The commonly used excipients and other active ingredients usually present in formulations did not interfere even if they were present in amount than they usually exist.

Analysis of formulations: Commercial formulations (tablets) containing 7-ADCA were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically with F and t tests and found not to different significantly. Percent recoveries were determined by adding standard drug to preanalyzed formulations. The results of the recovery experiments by the proposed methods are also listed in Table 3.

Chemistry of the colored species: Method Ma: Even though the first step (i.e oxidation of drug with NBS), different experimental conditions are maintained so as to suit the conditions for the second step (color development or decrease in the intensity of coloration corresponding to unreacted NBS). Insitu formed PMBQMI from PMAP and NBS involves in charge – transfer complex with sulphanilic acid (SAc) step (II). The probable sequences of reactions through analogy are presented in scheme 1.

Table 1a: Optimum conditions established for the proposed method M_a

Parameter	Optimum range	Conditions in procedure	Remarks
l_max(nm)	515-525	520
Effect of volume of (4.94x10^-3M) NBS required for oxidation	1.8-2.2 ml	2.0 ml	Addition of < 1.8 ml results in low absorbance particularly at higher concentration within the Beer’s law limits due to consumption of NBS. Addition of > 2.2 ml take the blank value beyond the detection limits of the instrument.
Time and temperature required for oxidation	15-20 min at lab temp	15 min at lab temp
Effect of volume of (8.75 x 10^-1M) acetic acid	0.25 - 0.75ml	0.5 ml	To fasten the oxidation step and maintain the pH at 3.0 ± 0.1 in color development step, 0.5 ml of acetic acid was found necessary.
Effect of volume of (8.71 x 10^-3M) PMAP solution	1.0 - 2.0ml	1.5 ml	< 1.0 and > 2 ml of PMAP solution produced low absorbance and high blank values respectively.
Keeping time	10-20 min	15 min	A minimum period of time, 10 min was necessary for PMAP to undergo oxidation and beyond 20 min the quinoneimine formed in situ undergoes slow hydrolysis to quinone, which resulted in low sensitivity.
Effect of volume of (1.16 x 10^-2M) SAc solution	1.8 – 2.2 ml	2.0 ml	A minimum amount of 1.8 ml of SAc solution was necessary for CT complex formation with PMBQMI formed in situ.
Solvent for final dilution	Distilled water	Distilled water	Final dilution with other miscible solvents (methanol, acetone, acetonitrile) did not enhance the intensity of colored species; water is sufficient for final dilution.
Stability period after final dilution	Immediate 50 min	5 min	After the stability period, the intensity of the colored species was found to decrease with time.

Table 1b: Optimum conditions established for the proposed method M_b

Parameter	Optimum range	Conditions in procedure	Remarks
l_max(nm)	545-555	550	--
Effect of buffer p^H on color development	6.5-7.5 ml	7.0 ml	Variation of volume of buffer pH-7.0 beyond the lower and upper limits resulted in low absorbance values.
Volume of buffer required for maximum intensity of color	14.5-15.5 ml buffer	15.0 ml buffer	Variation of volume of buffer pH=7.0 beyond the lower and upper limits resulted in low absorbance values.
Effect of volume of 6.60x10^‑3M Haet 1.42x10^-2M CAT.	0.5-1.2 ml for Haet 0.8-1.4ml for CAT	1.0 ml each for Haet, CAT	1.0ml of each of the two reagents was necessary for covering the broad range of Beer’s law limits.
Time (in buffer) and temp	15-20 min 65-75⁰C	2 min 70⁰C	The initial time of keeping of haematoxylin and CAT in buffer is 20 min the total temperature of this reaction is found 70⁰C
Stability period after final color	Immediate – 30 min	5 min	The stability period of the colored species is 30 min. Afterwards absorbance has gradually lowered with time at the rate of 1.5% for 5 min.

Table 1c: Optimum conditions established for the proposed method M_c

Parameter	Optimum range	Conditions in procedure	Remarks
l_max(nm)	535-545	540
Acid concentration (over all)	0.15-0.35 M HCl 0.10-0.25M H₂SO₄ 0.3-0.5M ACOH	0.25 M HCl	The studies on the variation of acid concentration indicated that constant absorbance value was obtained in 0.15-0.35M HCI, 0.10 to 0.25M H₂SO₄ or 0.3-0.5M ACOH at NBS concentration of 200 mg. As the difference in absorbance between the sample and the blank was found to be higher for the HCI medium, subsequent studies were carried out in 0.25M HCI.
Amount of NBS (100 mg.ml^-1) and amount of CB 50mg ml^-1	1.0-4.0 ml of NBS 8.0-12.0 ml of CB	2.5 ml of NBS 10.0ml of CB	In order to ascertain the linear relationship between the concentration of added NBS and the corresponding decrease in the absorbance of CB, experiments were carried out in 0.25M HCI mediums with varying amounts of NBS. As the decrease in absorbance was found to be linear up to an amount of 250 mg (2.5ml of 100 mg. ml^-1) of NBS, subsequent studies were carried out with 500 mg (10.0ml of 50 mg.ml^-1) of CB and 250 mg of NBS in 0.25M HCI mediums.
Time and temperature for oxidation with NBS	5-20 min at lab temp (28±5⁰C)	10 min	10 min waiting period at laboratory temperature (28±5⁰C) was adequate in oxidation stage of 7-ADCA. Variation of oxidation time beyond the upper and lower limits results in low absorbance values.
Time for oxidation of dye and stability period of final color	2-30 min	5 min	A minimum of 2 min was required for maximum color difference, which remained stable for 30 min. The difference in absorbance varied slowly and steadily beyond 60 min.

Table 2.Optical and regression characteristics, precision and accuracy of the proposed methods for 7-ADCA

Parameter	M_a	M_b	M_c
l_max (nm)	560	630	520
Beer’s law limits (mg/ml)	4 – 24	8 – 48	8 – 48
Detection limit (mg/m)	2.093	1.001	1.316
Molar absorptivity (L.mol/cm)	7.205 x 10³	5.404 x 10³	2.899 x 10³
Sandell’s sensitivity (mg/cm²/0.001 absorbance unit)	1.550 x 10^-1	0.1959	0.2937
Optimum photometric range (mg/ml)	12-20	24 - 48	20 – 48
Regression equation (Y=a+bc) slope (b)	0.02075	0.0127	0.0130
Standard deviation on slope (S_b)	7.901 x 10^-4	1.440 x 10^-4	9.852 x 10^-5
Intercept (a)	5.5 x 10^-3	1.249 x 10^-3	6.999 x 10^-3
Standard deviation on intercept (S_a)	10.48 x 10^-3	3.821 x 10^-3	2.614 x 10^-3
Standard error on estimation (S_e)	9.994 x 10^-3	3.644 x 10^-3	2.492 x 10^-3
Correlation coefficient (r)	0.9966	0.9998	0.9996
Relative standard deviation (%)*	1.222	0.8248	1.542
% Range of error (confidence limits)
0.05 level	1.405	0.9484	1.773
0.01 level	2.203	1.4872	2.780
% error in Bulk samples **	-0.241	0.166	0.282

*average of three determinations ** Average of six determinations

Table 3: Assay of 7-ADCA in Pharmaceutical Formulations

Formulations*	Amount taken (mg)	Amount found by proposed Methods**				Percentage recovery by proposed methods***
Formulations*	Amount taken (mg)	M_a	M_b	M_c	Reference method	M_a	M_b	M_c
Tablet I	60	59.56±0.53 F=2.393 t=1.257	59.63±0.60 F=1.86 t=1.024	59.72±0.65 F=1.59 t=0.777	60.05±0.82	99.83±0.99	99.62±0.76	99.90±0.95
Tablet II	60	59.58±0.60 F=1.604 t=1.222	59.62±0.62 F=1.502 t=1.104	59.43±0.58 F=1.717 t=1.628	60.04±0.7	99.31±0.93	99.66±0.55	99.46±0.82
Tablet III	60	59.42±0.58 F=2.302 t=1.54	59.63±0.67 F=1.725 t=0.983	59.33±0.70 F=1.5804 t=0.964	60.06±0.8	99.90±0.32	99.63±0.98	99.94±0.73
Tablet IV	60	59.32±0.60 F=2.025 t=1.755	59.47±0.58 F=2.167 t=1.427	59.71±0.59 F=2.326 t=0.8601	60.08±0.9	99.54±0.60	99.68±0.98	99.86±0.65

^*Tablets from four different pharmaceutical companies. ^**Average ± standard deviation of six determinations, the t-and F-test values refer to comparison of the proposed method with the reference method. Theoretical values at 95% confidence limit, F = 5.05, t = 2.57; ***Recovery of 10 mg added to the pre-analyzed pharmaceutical formulations (average of three determinations)

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3. Yamazaki T, Tsuchiya K, 3-Deacetoxy-7-(alpha-amino-1-cyclohexenylacetamido) cephalosporanic acid (SCE-100), a new semisynthetic cephalosporin III. Comparative studies on absorption, distribution and excretion of SCE-100 and cephalexin (CEX) in laboratory animals. J Antibiot (Tokyo). 1976; 29(5): 571-8.

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10. Jette

Thykaer, Bjarke Christensen,Jens Nielsen. Metabolic Network Analysis of an Adipoyl 7-ADCA-Producing Strain of Penicillium chrysogenum: Elucidation of Adipate Degradation: Metabolic Engineering. 2002; 4 (2): 151-158

11. Aki, Kanji, Tsuchiya, 3-deacetoxy-7-(α-amino-1-cyclohexenylacetamido) cephalosporanic acid (sce-100), A new semisynthetic cephalosporin I, comparative in vitro antibacterial activities of sce-100 and cephalexin (cex). Toshiyuki. The Journal of Antibiotics. 1976; 29(5): 559-565

12. Dutta N, Monali Dutta Saikia, Adsorption equilibrium of 7-aminodeacetoxy cephalosporanic acid–cephalexin mixture onto activated carbon and polymeric resins. Indian Journal of Chemical Technology. 2005; 12: 296-303

13. Medikondu Kishore, A.Koteswarao, M.Janardhan, New Spectrophotometric Methods for Quantitative Determination of 7-ADCA in Pharmaceutical Formulations, International Journal of Pharma Sciences and Research (IJPSR) Vol.1(8), 2010, 312-319

INTRODUCTION:

Parameter

CONCLUSIONS:

Table 3: Assay of 7-ADCA in Pharmaceutical Formulations

Amount found by proposed Methods**

Ma

Ma

Tablet I

M_a

M_a