EVALUATION OF METROLOGICAL CHARACTERISTICS OF SPECTROPHOTOMETRIC QUANTITATIVE DETERMINATION OF PARACETAMOL IN TABLETS BY SPECIFIC ABSORBANCE

The validation characteristics of the spectrophotometric quantitative determination of paracetamol in tablets by specific absorbance according to the British Pharmacopoeia (BPh) have been evaluated. The results of paracetamol content of 83.59% and 84.39% in terms of the average mass of one tablet do not meet the permissible limits В ±5.0%. The peculiarities of the sample preparation method for quantitative determination of the active pharmaceutical ingredient in tablets has been discussed, and comparative analysis of “Dissolution” and “Assay” tests for paracetamol tablets according to the BPh has been conducted. We have suggested to make such changes at the stage of the sample preparation as “... place the flask in an ultrasonic bath for 30 min...” instead of “...shake for 15 minutes...”. The acceptance criteria of the assay method for paracetamol tablets have been calculated for permissible limits of ±5.0%, ±7.5%, ±10.0%. The results of the convergence and linearity research of the method meet requirements for the permissible limits of ±5.0%. The results of the intermediate precision research of the method meet requirements for the permissible limits of ±7.5%. The results of the accuracy research of the method meet requirements for the permissible limits of ±10.0%. Taking into account the technical capabilities of the Ukrainian producers and a diverse list of excipients used in the manufacture of the drug, the spectrophotometric quantitative determination of paracetamol tablets by specific absorbance is recommended to use with the permissible limits of ±10.0%. The prognosis of the total uncertainty of the analysis results is consistent with requirements to the maximum permissible uncertainty of the analysis = 2.6 ≤ max = 3.2% and with results of the 3rd round of the Professional Testing Programme (PTP) of “Pharma-test” laboratories in the system of the State Inspection for Medication Quality Control of the Ministry of Public Health of Ukraine.

Paracetamol belongs to the group of non-steroidal anti-inflammatory drugs, it is a nonselective COX inhibitor, and over 50 years it has already been used as an antipyretic and analgesic [10]. Monocomponent formulations based on paracetamol tablets, capsules, solutions, suppositories, suspensions, granules, gel are produced by pharmaceutical industry. Paracetamol is part of many combined medicines with antipyretic and analgesic effects. The research concerning the use of paracetamol to treat pain in neonates as an alternative to opiates is being performed [12].
Quantitative determination of paracetamol in the substance according to the monographs of the State Pharmacopoeia of Ukraine (SPhU) [7], European [11], British [9] pharmacopoeias and Pharmacopoeia of the Republic of Belarus [3] is carried out by the ceriometry method, American [16], Japan [14] Korea [15] pharmacopoeias by the spectrophotometric method (by standard), China [13] -by specific absorbance.
Thanks to the introduction of quality assurance systems for results of analysis, equipment qualification the specific absorbance method has been widely used in phar-macopoeial analysis. At present the specific absorbance method is recommended by the SPhU not only for quantitative determination of 10 substances [7], but also for 21 types of medicinal plants [6]. A standardized procedure of validation of spectrophotometric methods for quantitative determination of drugs by specific absorbance has been developed [4] and successfully approved on the quantitative determination methods for prednisolone and riboflavin substances [18].
The aim of this research is to evaluate the metrological characteristics of spectrophotometric quantitative determination of paracetamol in tablets by specific absorbance, which is recommended by the British Pharmacopoeia (BPh) and to determine acceptable permissible limits for this method.

Experimental Part
Tablets "Paracetamol", 200 mg, manufactured by the pharmaceutical company "Darnitsya", batch UA / 4369/01/01 were chosen as an object of the research.
The following analytical equipment was used: a "SPE-CORD 200" spectrophotometer, AV 204 S / A METTLER TOLEDO analytical balance. Reagents, measuring glassware of class A (first class) and excipients meeting the requirements of the SPhU were used for the work.
The assay method for paracetamol in tablets according to the British Pharmacopoeia [9]: weigh and powder 20 tablets.
Add an accurately weighed powder containing 0.15 g of paracetamol to 50 ml of 0.1 M sodium hydroxide, dilute with 100 ml of water, shake for 15 minutes and dilute to 200 ml with a sufficient amount of water. Mix, filter and dilute 10 ml of the filtrate to 100 ml with water. Add 10 ml of the solution obtained to 10 ml of 0.1M sodium hydroxide, dilute to 100 ml with water and measure the absorbance of the solution obtained at the maximum at 257 nm. Calculate the content of C 8 H 9 NO 2 taking 715 as the value of A ( ) at the maximum at 257 nm. The nominal content of paracetamol b nom is 200 mg; the average weight of one tablet is 256.02 mg. The content of paracetamol in one tablet in terms of the average weight of one tablet in percentage of the prescribed amount was calculated by the formula: where: D -is dilution of the sample analyzed, m -is the mass of the sample for analysis. In our case, dilution is:

Results and Discussion
According to the specific absorbance method it is possible to obtain the correct results using a high level of equipment, its qualification and compliance with the requirements of the SPhU [7]. Taking this into account the qualification spectrophotometer characteristics were evaluated before the experiment. The control of cells, absorbance accuracy, absorbance convergence with removing cells, the limit of stray light have been carried out. The results obtained meet requirements of the SPhU.
The acceptance criteria of the assay method for paracetamol tablets was calculated considering the peculiarities of spectrophotometry by the specific absorbance method [4] for permissible limits of 95-105% (B = ±5.0%) and ±7.5%, ±10.0% according to the monograph (Tab. 1).
At first quantitative determination of paracetamol tablets in the concentration of 100% in accordance with the prescribed amount was carried out. To control correctness of the results and accuracy of the sample preparation two parallel studies of the tablet powder were conducted. Immediately after preparing analytical solutions according to the method, absorbance (A) was measured at the absorbance maximum of 257 nm three times with removing the cells. The results of the paracetamol content of 83.59% and 84.39% in terms of the average mass of one tablet do not meet the permissible limits (Tab. 2).
According to the results of the accuracy control of the sample preparation |Х 1 -Х 2 | = 0.80% < Δ As 1.60%; the negative result cannot be associated with the analyst's errors.
Peculiarities of the sample preparation of quantitative determination methods for the active pharmaceutical ingredient (API) in tablets. Determination of the quantitative content of the API in tablets has certain features that must be considered in standardization of methods. An accurately weighed quantity is dissolved in a suitable solvent in one or several steps using measuring glassware in quantitative determination of substances. Each step of the sample preparation is a part of uncertainty, which is calculated from the values of permissible uncertainty of measuring glassware and weighing according to the SPhU. In addition to the abovementioned sample preparation steps the method includes such additional operations as weighing of 20 tablets, powdering, dissolving and filtering, which bring more uncertainty to the total uncertainty of the sample preparation in quantitative determination of the API in tablets.
The relationship of "Dissolution" and "Assay" tests for paracetamol tablets according to the BPh. It should be noted that according to the BPh monograph control of dissolution of paracetamol tablets and "Assay" test for the API in tablets are carried out by UV-spectrophotometry using specific absorbance [9]. "Dissolution" and "Assay" tests are quite similar in operations, but differ in terms of dissolution, the purpose and test evaluation.
"Dissolution" test determines the minimum quality requirements for pharmaco-technological properties of paracetamol tablets regardless of the manufacturer (the composition of excipients, technology (Tab. 3)) based on the API quantitative determination after dissolution.
Conditions for "Dissolution" test: place one tablet in Apparatus II (paddle apparatus), rotate the paddle at 50 rpm (tolerance ±4%); the medium is phosphate buffer, pH 5.8 (±0.05 units), carry out dissolution at a temperature from 36.5° to 37.5°C; assess the API release in 45 minutes; dilute 20 ml of the filtrate with 0.1M sodium hydroxide to the concentration of 0.00075% (w/v); measure the absorbance of this solution; the amount of Table 1 The critical values of the systematic error (max δ tot ), total uncertainty of the analysis (maxΔ As ) and parameters of the linear dependence Y i = b•X i + a* the active ingredient in the solution should be not less than 70% of the prescribed amount.
Conditions for "Assay" test: add an accurately weighed quantity of the powder to 50 ml of 0.1 M sodium hydroxide, dilute with 100 ml of water, shake for 15 minutes; dilute 10 ml of the filtrate with 0.1 M sodium hydroxide to the concentration of 0.00075% (w/v); measure the absorbance of this solution; the amount of the active ingredient in the solution should be within the range of 95%-105% of the prescribed amount.
The question is if the API of paracetamol can be completely released under the following conditions for 15 minutes. Paracetamol belongs to the 1 st class of the biopharmaceutical classification system (BCS) and is considered to be very instant (at least 85% of the prescribed amount of the API passes into the solution for 15 min when using the paddle apparatus (50 or 75 rpm) or basket apparatus (100 rpm)) [2, 17]. The quality of 17 batches of 10 names of paracetamol tablets made in Russia and Western Europe was comparatively assessed in terms of the content of the API and the rate of dissolution (quantitative determination of the API for each batch was performed by HPLC (n = 10) according to the EuPh monograph "Paracetamol"). The results show that in 30 min 44.0±3.3% of the API of paracetamol was released for one batch; for 6 batches the dissolution percentage was in the range of 88.0±1.3% -94.0±1.1%; for 10 batches it was 96.0±0.7% -100.0±0.4% [1]. Thus, the difference in the release of the API may be associated with the composition of the excipients of tablets and their different formulations that each manufacturer sets independently.
Considering the facts described above the stage of the sample preparation of the method -"…shake for 15 mi-nutes…" should be changed to "… place the flask in an ultrasonic bath for 30 min…". Two parallel experiments were conducted with the tablet powder. The results concerning the paracetamol content in terms of the average mass of one tablet of 97.74% and 98.03% meet the permissible limits (Tab. 2). Thus, too low results ob- Table 2 The results of the spectrophotometric quantitative determination of paracetamol tablets by specific absorbance  Table 3 Comparative analysis of the excipients when producing paracetamol tablets by the Ukrainian manufacturers Further research and evaluation of validation characteristics of quantitative determination methods for paracetamol tablets by specific absorbance (the sample preparation with changes) were performed according to the standardized procedure of validation of spectrophotometric methods of quantitative determination of drugs by specific absorbance [4].

The prognosis of the total uncertainty of the analysis results (Δ As )
The prognosis of uncertainty of the sample preparation. The approach and requirements for maximum permissible errors for volumetric glassware, balances and devices were used to assess uncertainty of the sample preparation [7, 8]: The prognosis of the total uncertainty of the analysis result for the permissible limits of ±5.0%; ±7.5%; ±10.0% was conducted according to the standardized procedure of validation of spectrophotometric methods by specific absorbance [4]: The total uncertainty should be insignificant compared with the maximum permissible uncertainty of the analysis results: The total uncertainty of the analysis results exceeds the maximum permissible uncertainty for the permissible limits of ±5.0% and meets requirements for the permissible limits of ±7.5% and ±10.0%.
Accuracy, linearity, repeatability, intermediate precision were investigated using 9 model solutions within the whole range of the method application from 80 to 120% of the prescribed amount. The assessment of linearity was performed in the normalized coordinate system (Fig. 1). The results are shown in Tab. 4. The Table shows that the requirements for the parameters of the linear dependence are performed for permissible limits of ±5.0%.
The assessment of the validation parameters of the method is given in Tab. 5. Parameters of the accuracy and convergence are shown graphically in Fig. 2. The results of the convergence research of the method meet requirements for the permissible limits of ±5.0%. The results of the intermediate precision research of the method meet requirements for the permissible limits of ±7.5%. The results of the accuracy research of the method meet requirements for the permissible limits of ±10.0%. In this case without the other tests results (e.g. Art. 2.9.3. "Dissolution", Art. 2.9.6. "Uniformity of the content of the active ingredient per unit dosage of a medicinal pro-  duct") conclusions about the quality of the tablets cannot be done. Taking into account the technical capabilities of the Ukrainian producers and a diverse list of excipients used in the manufacture of the drug, the spectrophotometric quantitative determination of paracetamol tablets by specific absorbance is recommended to use for quantitative determination of the API in the drug with the permissible limits of ±10.0%, while the permis-sible limits of ±7.5% results may be doubtful. The prognosis of the total uncertainty of the analysis results is consistent with requirements to the maximum permissible uncertainty of the analysis = 2.6 ≤ max 3.2% and with results of the 3rd round of the Professional Testing Programme (PTP) of "Pharma-test" laboratories in the system of the State Inspection for Medication Quality Control of the Ministry of Public Health of Ukraine [5]. CONCLUSIONS The validation characteristics of the spectrophotometric quantitative determination of paracetamol in tablets by specific absorbance according to the British Pharmacopoeia have been evaluated. We have suggested to make such changes at the stage of the sample preparation as "… place the flask in an ultrasonic bath for 30 min…" instead of "…shake for 15 minutes…". Taking into account the technical capabilities of the Ukrainian producers and a diverse list of excipients used in the manufacture of the drug, the spectrophotometric quantitative determination of paracetamol tablets by specific absorbance is recommended to use for quantitative determination of API in the drug with the permissible limits of ±10.0%.  The results of the accuracy and convergence research of the spectrophotometric quantitative determination of paracetamol tablets by specific absorbance