Development And Validation of a Spectrofluorimetric Method for The Estimation of Doxycycline in Bulk and Formulation

Doxycycline, a member of the tetracycline class of antibiotics, plays a pivotal role in both clinical and pharmaceutical arenas. Recognized for its broad-spectrum antimicrobial properties, Doxycycline is widely prescribed for the treatment of various bacterial infections, including respiratory tract infections, skin infections, and certain sexually transmitted diseases. The mechanism of action of Doxycycline involves inhibiting bacterial protein synthesis by binding to the 30S ribosomal subunit, thereby impeding the attachment of aminoacyl-tRNA to the mRNA-ribosome complex. This interference halts the progression of bacterial protein synthesis, ultimately inhibiting bacterial growth and reproduction. Beyond its therapeutic applications, Doxycycline is a subject of interest in pharmaceutical research, particularly in the development of analytical methods for its quantification. Researchers leverage its distinct chemical properties to establish accurate and precise analytical techniques, ensuring the reliable assessment of Doxycycline concentrations in pharmaceutical formulations. The IUPAC name of Doxycycline is (4S,4aR,5S,5aR,6R,12aS)-4-(Dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide. In the context of pharmaceutical research, a focus on the physico-chemical properties of Doxycycline is crucial for the development and validation of analytical methods. Spectroscopic techniques, chromatography, and other analytical approaches are commonly employed to accurately quantify Doxycycline in pharmaceutical formulations. These methods ensure the quality, safety, and efficacy of the antibiotic, aligning with the rigorous standards of pharmaceutical research and development. As with any medication, the utilization of Doxycycline should be guided by healthcare professionals to ensure proper dosing, monitoring, and awareness of potential side effects or interactions. In the research setting, the precise application of analytical methods is essential to harness the full potential of Doxycycline in pharmaceutical formulations and contribute to advancements in antibiotic therapy.  Considering the physico-chemical as well as pharmaceutical properties of Doxycycline, an accurate, precise and cost-effective spectrofluorometric method was developed and validated. Developed method was successfully used for the estimation of Doxycycline in the marketed formulation of Doxycycline i.e., Doxycycline Tablets IP 100mg (Lexicare Pharma Pvt. Ltd.).

Fig.1. Chemical structure of Doxycycline

MATERIALS AND METHOD

Material:

Doxycycline has been purchased from TCI Chemicals (India) Pvt. Ltd, Chennai. Methanol was purchased from Merck. All the chemicals of analytical grade were used for the proposed study.

Instruments used:

The spectrofluorimetric study was carried out with a Shimadzu RF-5301 fluorimeter to determine levels of fluorescence in the Doxycycline. A Xenon 150w lamp was used as a light source. Quartz cells having 48mm height, 10mm path length with 0.5mm slit width were used for fluorescence measurement. Weighing balance (Vibra HT, Essae) with internal calibration mode was used for the weighing purpose.

Preliminary analysis:

A preliminary analysis was carried out to determine the excitation and emission wavelength of Doxycycline. Various solvents like distilled water, methanol, acetonitrile and their combinations were used to determine appropriate media for Doxycycline. Doxycycline showed maximum fluorescence intensity in Methanol: Water (40:60%v/v) as a media. Initially, Doxycycline solution of 100ng/ml strength was prepared in methanol. Prepared solution was scanned spectrofluorimetrically to obtain the excitation and emission wavelengths. The scanning was performed over 220 nm to 600 nm range and excitation and emission wavelength were found to be 354nm and 395nm (figure.2) respectively.

Fig. 2: Excitation and Emission Spectra of Doxycycline

Accurately weighed 5 mg of Doxycycline was transferred into the calibrated volumetric flask and dissolved in 10 ml water to achieve a stock solution of 1000 μg/ml (stock-I). Stock- I solution was suitably diluted with water to achieved further calibration standards.

Construction of Calibration Curve:

Calibration curve was prepared by diluting the stock-I (1000 µg/ml) solution to achieve the seven different calibration standards representing CAL STD 1 (50ng/ml), CAL STD 2 (100ng/ml), CAL STD 3 (200ng/ml), CAL STD 4 (300ng/ml), CAL STD 5 (600ng/ml), CAL STD 6 (900ng/ml), CAL STD 7 (1000ng/ml) strength. The fluorescence intensity was measured at pre-defined excitation and emission wavelengths of 354 and 395 nm respectively. The calibration curve representing concentration vs. Fluorescence intensity was plotted using excel program of Microsoft office 2013. Above mentioned procedure was repeated three times, so that reproducible results can be obtained.

Spectrofluorimetric Method Validation:

Validation is the process which provides a high degree of assurance, so as to produce a desired result and meeting its predetermined specifications and quality characteristics. Developed fluorimetry method for the estimation of Doxycycline was validated as per the ICH guidelines. Different validation parameters like linearity and range, accuracy, precision, robustness, ruggedness, limit of detection (LOD) and limit of quantitation (LOQ) were calculated using predefined calibration standards and or quality control standards as described below ^[11-12].

Linearity and Range:

Linearity of the proposed spectrofluorimetric method was calculated by using seven different calibration standards. After analysis of calibration standards, calibration curves in terms of Concentration vs. Fluorescence intensity plots were developed and subjected to linear least square regression analysis. R² value was considered to be important factor for determining the linearity of the proposed method.

Accuracy:

To determine the accuracy of the method, different quality control solutions were prepared independently from stock-I i.e., LQC: 60ng/ml, MQC: 450ng/ml and HQC: 950ng/ml and analyzed at level of 80%, 100% and 120% of its predefined concentrations to the predefined concentrations, different amounts of Doxycycline were added (standard addition method) and the accuracy was calculated on the basis of percent recovery.

Precision:

The precision of the method was checked by preparing different quality control solutions independently from stock-I i.e., LQC: 60ng/ml, MQC: 450ng/ml and HQC: 950ng/ml at three different time intervals in a day. Same procedure was followed on three different consecutive days so as to obtain inter-day variation. The fluorescence intensities for Doxycycline were recorded and the results were expressed as % Relative Standard Deviation (%RSD).

Robustness:

Robustness of the proposed spectrofluorimetric method was established by changing composition of the ethanol by ± 1.0 %. MQC samples of Doxycycline were prepared in methanol with and analysed at 354nm and 395nm (excitation-emission wavelength of Doxycycline). The results were calculated in terms of % RSD.

Ruggedness:

Ruggedness of the proposed method was studied by analyst variation. MQC samples of Doxycycline were prepared by three different analysts of the laboratory and were analyzed at 354nm and 395nm. The results were calculated in terms of % RSD.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and the LOQ of the drug were calculated by using the following equations as per ICH guidelines.

LOD = 3.3 × SD/S

LOQ = 10 × SD/S

Where, SD= standard deviation of lower most concentration of calibration curve S= Slope of calibration curve.

Estimation of Doxycycline in Bulk and Marketed Formulation:

The Doxycycline content in its marketed formulation (Doxycycline Tablets 100mg - Lexicare Pharma Pvt. Ltd.) was estimated using pre-validated UV-Visible spectrophotometric method. Tablet formulation contents (labeled strength: 100 mg/tablet) were dissolved in 1 ml of co-solvent system to achieve a stock solution of 1500 ng/ml (n=5). Said solution was suitably diluted with co-solvent system and analysed for the Doxycycline content using proposed spectrofluorimetric method.

RESULTS AND DISCUSSION

Construction of Calibration Curve:

Quantification of Doxycycline samples by any instrumental method of analysis needs a reproducible calibration curve and a mathematical equation stating correlation between concentration and the response. As compare to graphical method, above stated method is widely accepted and reproducible in nature. To establish linearity of the proposed method, seven different calibration standards were prepared from the stock solution and analyzed at excitation wavelength 354nm and emission wavelength 395nm by spectrofluorimeter. Least square linear regression analysis was performed for the obtained spectrofluorimetric data using MS-excel 2013. Calibration curve was repeated five times for reproducibility. Various concentrations and their fluorescence intensities with mean ± standard deviation was reported (Table 1).

Table 1: Calibration Standard Data for Doxycycline

Spectrofluorimetric Method Validation

Linearity and range:

Linearity and range are the important parameters of analytical method that demonstrates the limit within which the intended method is to be used for its optimum performance. Considering the prime importance of linearity and the range, seven-point calibration curve of Doxycycline was plotted covering a range of 50-1000 ng/ml. Different concentrations and the respective mean fluorescence intensities values are depicted in table 1. Calibration curve when subjected to least square regression analysis yielded an equation; y = 0.9098x+2.0298 with correlation coefficient 0.9999 (figure 3). From the linearity study, it was revealed that, developed method was linear over the concentration range of 50 to 1000ng/ml.

Fig. 3 Calibration Curve for Doxycycline

Accuracy is the closeness of test results to the true value obtained by method. The accuracy of an analytical method should be established over its calibration range so that at any point of determination, results obtained would be accurate. For Doxycycline, accuracy was determined using recovery studies. At 80%, 100% and 120% standard addition, mean recovery of Doxycycline was found in between 99.26% to 100.19%. The relative standard deviation (% RSD) was found to be less than 2 as shown in table 2. From the results of accuracy studies, it was predicted that developed method is highly accurate.

Precision is defined as closeness of agreement among the individual test result when the method is applied repeatedly to multiple sampling of homogeneous sample. Precise analytical method leads to accurate results. Intra-day and inter-day precision of spectrofluorimetric method was established at LQC: 60ng/ml, MQC: 500ng/ml and HQC: 950ng/ml levels of Doxycycline. The results expressed in terms of mean fluorescence intensity values, % assay and % RSD for the intra-day and inter-day precision study are demonstrated in table 3 and table 4 respectively. Percent RSD values of intra-day precision study were found to be in between 0.335 and 1.500, whereas those of inter-day precision study were in between 0.107 and 1.249 overall; % RSD values of less than 2 demonstrated that developed spectrofluorimetric method is precise and reproducible.

Table 4: Inter-Day Precision Data of Spectrofluorimetric Method for Doxycycline