Determination of Doxycycline in Doxycycline Hydrochloride Soluble Powder under Different Chromatographic Conditions

Lanying GUO Youjiang DIAO Shijin GUO

Abstract ［Objectives］This study was conducted to compare the accuracy and detection speed of high performance liquid chromatography for determining the content of doxycycline hydrochloride in doxycycline hydrochloride soluble powder under different chromatographic conditions, in order to improve the level of laboratory testing.

［Methods］ Four sets of experiments were designed through a cross-test method. The contents of doxycycline hydrochloride in the raw materials of doxycycline hydrochloride and doxycycline hydrochloride soluble powder were determined by changing the chromatographic column and mobile phase conditions, and the feasibility and practicability of the four methods were judged by comparing the detection results through data and chromatographic signal processing.

［Results］ The content of doxycycline hydrochloride in finished doxycycline hydrochloride soluble powder and the raw materials of doxycycline hydrochloride could be accurately determined under the four chromatographic conditions, of which the experimental group using Agilent C18 column and the mobile chromatography conditions of finished doxycycline hydrochloride soluble powder had the shortest retention time.

［Conclusions］ The high performance liquid chromatography method using Agilent C18 chromatographic column and mobile phase chromatographic conditions of finished doxycycline hydrochloride soluble powder products can quickly and accurately determine the content of doxycycline hydrochloride in doxycycline hydrochloride soluble powder.

Key words Doxycycline hydrochloride; Chromatographic conditions; High performance liquid chromatography

Received: June 12, 2022  Accepted: August 22, 2022

Lanying GUO (1999-), female, P. R. China, major: veterinary medicine.

*Corresponding author. E-mail: gsj7877@126.com.

Doxycycline hydrochloride, also known as vibramycin, has a molecular weight of 480.896. It is a yellow or light yellow crystalline powder, which is odorless, bitter in taste, easily soluble in methanol and water, and the aqueous solution is unstable. The molecular structure contains phenolic hydroxyl group, enoyl and dimethylamino group. Doxycycline hydrochloride is an acid-base amphoteric compound, which is relatively stable in weakly acidic solution (pH 2-7)［1］.

Doxycycline hydrochloride belongs to the tetracycline class of antibiotics and is a fast-acting bacteriostatic agent. It mainly binds reversibly to the receptor on the 30S subunit of bacterial ribosome, interferes the formation of the ribosome complex from tRNA and mRNA, and prevents the elongation of peptide chains and thereby inhibits protein synthesis, and thus, the growth and reproduction of bacteria is rapidly inhibited［2］. Doxycycline can increase the permeability of the cell membrane of bacteria and other microorganisms, so that the important substances in cells are leaked out. In addition, doxycycline hydrochloride also has anti-inflammatory, antioxidant and other non-antibacterial effects［3］.

The antibacterial activity of doxycycline hydrochloride is better than that of tetracycline, oxytetracycline and chlortetracycline, 2 to 4 times that of tetracycline, oxytetracycline and chlortetracycline. It has the characteristics of broad-spectrum, potent and long-acting, no obvious toxicity to the kidney, and good antibacterial effects on microorganisms such as Gram-positive, negative bacteria, mycoplasma, chlamydia and rickettsia［2］. In veterinary clinics, it is often used for the prevention and treatment of respiratory tract infections caused by mycoplasma and chlamydia, as well as the prevention and treatment of diseases such as salmonellosis, colibacillosis, and brucellosis［4］. Doxycycline hydrochloride is often made into various formulations such as tablets, injections, injection powder, powder and capsules, among which the most common dosage form in veterinary drug production is doxycycline hydrochloride soluble powder. Doxycycline hydrochloride soluble powder is easily soluble in water, which is beneficial to group administration in intensive breeding production, so it is widely used in livestock and poultry breeding.

The conventional determination methods of doxycycline hydrochloride mainly include antibiotic microbiological assay, ultraviolet spectrophotometry and high performance liquid chromatography. The detection of doxycycline hydrochloride content by the antibiotic microbiological assay requires complex operation procedures and long detection time, which makes the quality control of the intermediate products in the production process not in place and leads to product quality problems［5］. Liu and Zhang［6］ compared the detection of doxycycline hydrochloride by UV spectrophotometry and high performance liquid chromatography, and concluded that ultraviolet spectrophotometry is convenient and quick, and high performance liquid chromatography is more reliable. Hu［7］ concluded through experiments that high-phase liquid chromatography is simple to operate, accurate in detection results, and can be used as a quality standard detection method.

In the new version of Veterinary Pharmacopoeia of the People’s Republic of China (2020 edition), high performance liquid chromatography was used to detect doxycycline hydrochloride soluble powder and the raw materials of doxycycline hydrochloride. Changes in mobile phase and column can affect the accuracy of the detection of drugs and the speed of detection and analysis.

This study aimed to compare the accuracy and detection speed of high performance liquid chromatography for the determination of doxycycline hydrochloride content in doxycycline hydrochloride soluble powder under different chromatographic conditions, in order to improve the laboratory detection level.

Materials and Methods

The experiment was conducted at Shandong Lvdu Ante Animal Veterinary Drug Industry February 2022 to May 2022.

Main instruments and agents

Liquid chromatograph: 1260 High Performance Liquid Chromatography Analyzer of Agilent Technologies, serial number: DEABB02952; column 1: nano-micro Unisil 5-120 C18 (4.6×250 nm, 5 μm); column 2: Agilent Eclipse XDB-C18 (4.6×250 mm, 5 μm); pH meter: Leici PHS-3EPH meter; ultrasonic cleaner: Scientz SB-3200DT; doxycycline hydrochloride standard: China National Institutes for Drug Control, Batch No.:210485-202002; doxycycline hydrochloride raw material: Hebei Jiupeng Pharmaceutical Co., Ltd., batch number: A202201071, and Yangzhou Lianbo Pharmaceutical Co., Ltd., batch number: YD210601118; doxycycline hydrochloride soluble powder: Shandong Lvdu Ante Animal Veterinary Drug Industry, batch numbers: 20220107, 20220304, 20220313; ammonia water: Yantai Shuangshuang Chemical Co., Ltd.; HCl solution: Yantai Yuandong Fine Chemical Co., Ltd.; glacial acetic acid (analytically pure): Yantai Shuangshuang Chemical Co., Ltd.; acetonitrile (chromatographic grade): Tianjin Kermel Chemical Reagent Co., Ltd.; ammonium acetate (analytically pure): Tianjin Tianli Chemical Reagent Co., Ltd.; diammonium hydrogen phosphate (analytically pure): Tianjin Bodi Chemical Co., Ltd.; triethylamine (analytically pure): Tianjin Fuyu Fine Chemical Co., Ltd.; N,N-dimethylformamide (chromatographic grade): Tianjin Tianli Chemical Reagent Co., Ltd.; disodium EDTA (analytically pure): Laiyang Shuangshuang Chemical Co., Ltd.; doxycycline reference substance, doxycycline hydrochloride, and doxycycline hydrochloride soluble powder, provided by Shandong Lvdu Ante Animal Veterinary Drug Industry, Binzhou Branch of Shandong Academy of Agricultural Sciences.

Chromatographic conditions and mass spectrometry conditions

The standards of chromatographic conditions referred to the conditions for the detection of doxycycline hydrochloride soluble powder and doxycycline hydrochloride in Veterinary Pharmacopoeia of the People’s Republic of China (2020 edition).

Chromatographic condition 1

Octadecylsilane-bonded silica gel was used as a filler for doxycycline hydrochloride chromatographic detection. The chromatography was carried out under following conditions: chromatographic column: C18 column, mobile phase［8］: acetate buffer solution［0.25 mol/L ammonium acetate solution-0.1 mol/L disodium EDTA-triethylamine (100∶10∶1), the pH of which was adjusted with glacial acetic acid or ammonia to 8.8］-acetonitrile (85∶15), column temperature: 35 ℃, detection wavelength: 280 nm, flow rate: 1.0 ml/min, and injection volume: 20 μl.

Chromatographic condition 2

Octadecylsilane-bonded silica gel was used as a filler for doxycycline hydrochloride chromatographic detection.  The chromatography was carried out under following conditions: chromatographic column: C18 column, mobile phase［9］: 0.05 mol/L ammonium oxalate-dimethylformamide-0.2 mol/L diammonium hydrogen phosphate (65∶30∶5), which was adjusted to pH 8.0±0.2 with ammonia test solution, isocratic elution, column temperature: 35 ℃, flow rate: 1 ml/min; detection wavelength: 280 nm, and injection volume: 20 μl.

Preparation of solutions

The concentration of hydrochloric acid was 0.01232/25×2/10×85.2%, and the test products were required to contain 0.1 mg per ml.

Preparation of mobile phase 1

According to the preparation ratio of the mobile phase, calculation was performed according to the formula C=nV→m=CMV. Accordingly, 9.635 g of ammonium acetate should be dissolved in 500 ml of water; 1.861 2 g of disodium EDTA was dissolved in 50 ml of water; and 5 ml of triethylamine was directly pipetted, and the three solutions were mixed. Then, 425 ml of solution was taken and measured with an electronic pH meter for pH, which was then adjusted. The pH meter was turned on, and the electrode was rinsed with pure water. When measuring pH, the glass membrane of the electrode should be completely submerged in the solution to be measured. The pH was adjusted with glacial acetic acid (i.e., acetic acid) or ammonia water, which was added dropwise without diluting slowly while shaking until the pH of the solution was adjusted to 8.8. After finishing, the electrode was rinsed with pure water. Next, 75 ml of acetonitrile was dissolved with the solution of pH 8.8, and the obtained solution was vacuum filtered. The filter paper of the suction filter was divided into two types, namely organic test paper and anhydrous test paper, which are distinguished by acetonitrile. The addition of acetonitrile to the organic test paper causes no obvious change, but the addition of acetonitrile to the anhydrous test paper will dissolve the anhydrous test paper. After vacuum filtration, the mobile phase was subjected to ultrasonic vibration at 25 ℃ for 15 min to remove air bubbles.

Preparation of mobile phase 2

First, 500 ml of mobile phase was prepared. According to the proportions, 325 ml of ammonium oxalate solution, 25 ml of diammonium hydrogen phosphate solution, and 150 ml of dimethylformamide solution should be prepared. According to the concentration calculation, 2.309 3 g of ammonium oxalate and 0.660 3 g of diammonium hydrogen phosphate should be accurately weighed. The pH of the solution was measured with an electronic pH meter and adjusted. The pH meter was turned on, and the electrode was rinsed with pure water. When measuring pH, the glass membrane of the electrode should be completely submerged in the solution to be measured. The pH was adjusted to 8.0 with ammonia solution, and the error should not exceed 0.2. The reagent was added dropwise directly and slowly without dilution while shaking. The solution was vacuum filtered. As no acetonitrile was contained, an aqueous membrane could be used. After vacuum filtration, the mobile phase was subjected to ultrasonic vibration at 25 ℃ for 15 min to remove air bubbles.

Preparation of reference solution

First, 0.012 5 g of the finished doxycycline hydrochloride soluble powder was precisely weighed, and added with 0.01 mol/L hydrochloric acid solution and diluted to a 25 ml volumetric flask, and the obtained solution was shaken well. Then, 2 ml of the solution was taken and then diluted to 10 ml, and after shaking well, a control solution containing 0.1 mg of doxycycline per ml was obtained for later use.

Preparation of test solutions

Doxycycline hydrochloride soluble powder (finished product) and doxycycline hydrochloride (raw materials) were accurately weighed, and added with 0.01 mol/L HCl solution for dissolution, and the mixtures were mixed well. They were quantitatively diluted to make a solution containing 0.1 mg of doxycycline per milliliter as the test solutions, and 20 μl of each test solution was injected into the liquid chromatograph to record the chromatogram. Appropriate amount of doxycycline reference substance was taken, and determined by the same method. According to the external standard method, the contents of doxycycline in the test samples were calculated by peak areas.

The preparation steps of doxycycline hydrochloride test solution were as follows:

① First, 0.06 g of the raw material powder was precisely weighed, and added in a 25 ml volumetric flask, and 0.01 mol/L HCl solution was added dropwise with shaking to the constant volume.

② Then, 1 ml of the test solution in step ① (after diluting to constant volume) was added into another 25 ml volumetric flask, and re-added with 0.01 mol/ml HCl solution to the mark, and the solution was shaken well to obtain a doxycycline hydrochloride test solution containing 0.1 mg/ml doxycycline hydrochloride.

③ The raw material test solution was prepared in parallel in two copies.

The preparation steps of doxycycline hydrochloride soluble powder test solution were as follows:

① First, 0.3 g of the finished powder was precisely weighed, and added into a 25 ml volumetric flask, and 0.01 mol/ml hydrochloric was added dropwise with shaking to the constant volume.

② Then, 2 ml of the test solution in step ① was added into another 25 ml volumetric flask, and re-added with 0.01 mol/ml HCl solution to the mark, and the solution was shaken well to obtain a doxycycline hydrochloride finished product test solution containing 0.1 mg/ml doxycycline hydrochloride.

③ The finished product test solution was prepared in parallel in two copies.

Precautions for the use of electronic balances:

① The weight reduction method was adopted when weighing. The bottle body and the medicine and medicine spoon were weighed together, and the tare weight was deducted. The absolute value of the medicine reading was taken as the actual weight of the medicine. The precise units for weighing raw materials and finished products were different, so electronic balances with different units should be used.

② The balance should be placed horizontally on a hard, smooth surface, indoors to prevent the light shining on the balance from directly affecting the accuracy of the balance. When weighing, the side door should be closed to reduce the error. The instrument will automatically calibrate by lightly pressing the tare key.

③ When the "→" mark appears in the lower left corner of the display, the number displayed on the display is the actual value of the weight of the substance being weighed.

④ After recording the data, the weighing paper or weighing bottle was taken out, and the side glass door was closed. The power should be cut off in time to end weighing.

Sample bottle preparation

The five groups of solutions were put into five injection bottles respectively (each bottle body was rinsed with corresponding solution in advance, and the solution entered the injection bottle after being filtered through a disposable syringe filter), and the bottle body was marked for ease of identification.

Application method of liquid chromatograph and matters needing attention

Application method

The parameters were set after power on. The mobile phase prepared in the above experiment was used, and whether the chromatographic column and mobile phase were correct was checked. This experiment was carried out in four groups, and the mobile phase and chromatographic column should be replaced and checked in time each time. The injection valve was flushed. First, the pipeline was pre-flushed with the mobile phase required for the experiment to replace the mobile phase in the pipeline to prevent the last residual mobile phase from affecting the accuracy of the experiment, and the injection needle was washed. The column was equilibrated, so as not to affect the accuracy of the experiment, and the sample running parameters could be set simultaneously. After the equipment was well equilibrated, a blank test was first performed to reduce system errors and eliminate system contamination. After the chromatographic column was stabilized, the tested solutions could be injected to run the test, and a report was issued after the end of each run.

Precautions

It is necessary to check the volume of the remaining solution of each mobile phase and its setting in time to prevent automatic pump stop due to the set volume being lower than the lower limit, which will affect the continuation of tests, and the pump washing solution should be added in time. During the liquid phase operation, it is necessary to check the working progress of the instrument from time to time, and timely and correctly handle the unexpected situation, so as not to affect the operation of the liquid phase.

Lanying GUO et al. Determination of Doxycycline in Doxycycline Hydrochloride Soluble Powder under Different Chromatographic Conditions

Experimental results

External standard method［10］: The reference substance and the test substances were accurately weighed (pipetted) according to corresponding regulations, and prepared into solutions. A certain amount was precisely pipetted, and injected, and the chromatogram was recorded. The peak area (or peak height) of the test product in the reference solution and the test solutions were measured, and the contents were calculated as follows:

Content (Cx)=CRAxAR

Cx is the concentration of the test substance; CR is the concentration of the reference substance; Ax is the peak area or peak height of the test substance; AR is the peak area or peak height of the reference substance.

A content Cx obtained in the range of 80%-120% was qualified.

Experimental data under column 1 and the mobile phase for raw materials

Data analysis

The contents of doxycycline hydrochloride in the finished doxycycline hydrochloride soluble powder products and raw materials detected under the above four chromatographic conditions were all within the qualified range and the relative deviation was small, indicating that the four groups of experimental data all had reference value. According to the SPSS method, the significance and sign two-tailed test results of the four groups of data were as follows:

The first group is the calculation results of the experimental data in "Experimental data under column 1 and the mobile phase for raw materials"; the second group was the calculation results of the experimental data in "Experimental data under column 1 and the mobile phase for finished products"; the third group was the calculation results of the experimental data in "Experimental data under column 2 and the mobile phase for raw materials"; and the fourth group was the calculation result of the experimental data in "Experimental data under column 2 and the mobile phase for finished products."

It can be seen from Table 5 that the calculation results of the significance and the sign two-tailed value between raw materials and standard products and between raw materials and finished products for the four groups of data were all within the specified range, indicating that  there were no differences in the test results of the raw materials of doxycycline hydrochloride and the standard products of doxycycline hydrochloride soluble powder and in the test results of the raw materials doxycycline hydrochloride and the finished doxycycline hydrochloride soluble powder products, which met the experimental requirements. In the same group of tests, the retention time of the finished doxycycline hydrochloride soluble powder and the raw materials of doxycycline hydrochloride powder were roughly the same, and the differences from the standard doxycycline hydrochloride soluble powder were within the allowable error range, indicating that the mobile phase for detecting the content of doxycycline hydrochloride in the finished doxycycline hydrochloride soluble powder can also be used to detect the raw materials of doxycycline hydrochloride. Similarly, the mobile phase for detecting the raw materials of doxycycline hydrochloride can also be used to detect the content of doxycycline hydrochloride in the finished doxycycline hydrochloride soluble powder. The peak area of finished doxycycline hydrochloride soluble powder was larger than that of the raw materials of doxycycline hydrochloride. When the chromatographic column was replaced, the retention time and peak area of finished doxycycline hydrochloride soluble powder and those of the raw materials of doxycycline hydrochloride changed, but the calculated doxycycline hydrochloride contents did not change significantly. In the fourth group of tests, Agilent C18 chromatographic column and mobile phase for the detection of doxycycline hydrochloride soluble powder products were selected. The test results of this group of experimental data were accurate and the time consumed was the shortest compared with other three groups of data. Therefore, it was concluded that Agilent C18 chromatography column and the mobile phase for the detection of finished doxycycline hydrochloride soluble powder products were the most rapid and concise for the detection of doxycycline hydrochloride in doxycycline hydrochloride soluble powder.

The above results showed that under the same chromatographic column conditions, the difference between the mobile phase for raw materials and the mobile phase for finished products was not obvious; and under the same mobile phase conditions, the retention time of Agilent C18 column was shorter than that of Nano-Micro C18 column.

Conclusions and Discussion

In this study, the mobile phases and chromatographic columns for the raw materials and finished products of doxycycline hydrochloride soluble powder recommended by the 2020 edition of Veterinary Pharmacopoeia of the People’s Republic of China were used to cross-test the finished products and raw materials. In the four groups of test data, the detection results of doxycycline hydrochloride in finished doxycycline hydrochloride soluble powder products (RSD 0.15%) were close to those of Jiang et al.［8］ (RSD 0.1%), and the test results of the raw materials of doxycycline hydrochloride (RSD 0.35%) were close to those of Lin et al.［11］ (RSD 0.49%). It showed that the mobile phase used to detect the doxycycline hydrochloride content in the finished doxycycline hydrochloride soluble powder could also be used to detect the raw materials of doxycycline hydrochloride. Similarly, the mobile phase used for detecting the raw materials of doxycycline hydrochloride could also be used for detecting the content of doxycycline hydrochloride in finished doxycycline hydrochloride soluble powder. The contents in doxycycline hydrochloride soluble powder and the raw materials of doxycycline hydrochloride were detected under the four chromatographic conditions, and the contents obtained according to the chromatographic data were all within the qualified range specified by Veterinary Pharmacopoeia of the People’s Republic of China (2020 edition)［12］. It showed that within the allowable range of error, the mobile phase for testing the raw materials of doxycycline hydrochloride can be used for detecting the content of doxycycline hydrochloride in finished doxycycline hydrochloride soluble powder. Similarly, the mobile phase for testing the content of doxycycline hydrochloride in finished doxycycline hydrochloride soluble powder can also be used for detecting the content in the raw materials of doxycycline hydrochloride. Lin and Sun et al.［11］ showed that the chromatographic conditions of doxycycline hydrochloride soluble powder could also be applied to the detection of other dosage forms such as doxycycline hydrochloride injection and doxycycline hydrochloride powder. This study showed that the cross-substitution of doxycycline hydrochloride raw materials and doxycycline hydrochloride soluble powder recommended by Veterinary Pharmacopoeia of the People’s Republic of China (2020 edition) did not affect the accuracy of doxycycline hydrochloride content detection and expanded the application scope of the LC detection method for the raw materials of doxycycline hydrochloride and doxycycline hydrochloride soluble powder.

In this study, under the same mobile phase conditions, the column was replaced, and it was found that the retention time changed significantly; and when the chromatographic column was fixed, the mobile phases for the raw materials and finished products of doxycycline hydrochloride were cross-replaced, and observation of the experimental data showed that the replacement of the mobile phase had little effect on the retention time. The research of Gao and Yan et al. showed that the change of retention time was related to the change of the physical parameters of the chromatographic column［13］. The results of this study confirmed the research conclusion of Gao and Yan et al.

The above study showed that the high performance liquid chromatography method using Agilent C18 chromatographic column and mobile phase chromatographic conditions of finished doxycycline hydrochloride soluble powder products can quickly and accurately determine the content of doxycycline hydrochloride in doxycycline hydrochloride soluble powder.

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