Hydrazine determination in allopurinol using derivatization and SPE for sample preparation
Graphical abstract
Introduction
During the synthesis of pharmaceuticals it occurs that genotoxic ingredients have to be used. In this case the amount of this compound must be limited at ppm level in the final product. This required concentration differs by 5–6 orders of magnitude from the amount of the API.
In order to determine an impurity at ppm level, a huge amount of sample is needed. This high concentration can decrease the efficacy of the analytical method; moreover the overload of the chromatographic system may occur. Therefore during the sample preparation the amount of the API needs to be decreased while the quantity should remain the same for the component to be determined.
Even if huge amount of a component is analyzed but the component has no significant UV absorption or cannot be separated properly from other impurities then appropriate analysis is not feasible. In such cases, derivatization reaction can solve the problem as it may change the solute properties which are then more favourable for its analysis.
The aim of this work was to determine hydrazine content in allopurinol API. Allopurinol is used for the treatment of patients with high uric acid level in blood (hyperuricaemia) since the drug inhibits an enzyme that participates in the formation of uric acid. The hyperuricaemia can result arthritis and in worse case kidney failure. Beside high serum uric acid level increases the risk of high blood pressure and heart failure [1]. Allopurinol drugs are on the market in 100 and 300 mg potency. During application for adults the recommended starting dose is 100 mg once a day. The daily dose can be raised as required (the blood uric acid level is checked in every 1–3 weeks) by 100 mg. The doctor defines the treatment dose based on the seriousness of the illness. In the case of mild hyperuricaemia 100–200 mg, in moderate state 300–600 mg and in serious state 700–900 mg is the suggested daily dose. According to the body weight the daily dose of the drug is 2–10 mg per body weight kilograms.
Fig. 1 shows one of the many synthesis routes for the production of allopurinol. The common of the routes is that every of them contain hydrazine as an important building block. Since hydrazine is genotoxic, it is a priority subject to determine its content during the quality control analysis of allopurinol.
Hydrazine is a corrosive and carcinogen substance. According to the European Medicine Agency (EMA) and International Conference on Harmonization (ICH) the maximum daily intake of potential genotoxic impurities for more than 12 months of exposure is 1.5 μg [3]. Considering the daily dose mentioned before, this means that the permitted hydrazine concentration in allopurinol is 2.5 ppm.
The European Pharmacopoeia (Ph.Eur.) contains a method for the determination of hydrazine content in allopurinol [2] but it requires lengthy sample preparation and the nowadays unpopular normal phase liquid chromatography.
The hydrazine molecule does not contain chromophore group so the commonly used UV–vis detector or the highly sensitive fluorescence detector cannot be applied for its direct detection. According to literature, titration [4], electrochemical [5] and spectrophotometric methods [[6], [7], [8]] are recommended for the determination of hydrazine. Furthermore liquid chromatographic method along with derivatization can be found where the samples were taken from biological medium [9] and environment [10].
The permitted concentration mentioned above is very low and accordingly the limit of detection must be fifth or tenth grade lower. According to spectrophotometric and liquid chromatographic literature, this concentration value can be achieved only by derivatization before injecting onto the chromatographic column. Seifart et al. [9] determined hydrazine at low concentration that meets the requirements but in our case the huge amount of matrix resulted in another problem. Several articles were published recently in which the amount of hydrazine was determined in APIs [[11], [12], [13]]. But in these cases the reduction of the amount of API wasn’t necessary during the sample preparation. Despite the fact that there is a big difference between the polarity of allopurinol and hydrazine still a huge amount of sample should be injected to the column to attain the low limit of detection. This could cause overloading and formation of new interactions which could destroy the selectivity of the column.
Section snippets
Experimental
The quality of the methanol (MeOH) was gradient grade. The quality of benzaldehyde used for the derivatization was reagent grade; the hydrazine sulfate and sodium hydroxide (NaOH) were analytical grade (Merck, Darmstadt, Germany). The allopurinol originates from the synthesis of Egis Pharmaceuticals Plc. Water was prepared freshly using ELGA Purelab system (ELGA, Lane End, UK).
Mettler Toledo analytical and precision balances were used for weighing (Greifensee, Switzerland). Eppendorf automatic
Derivatization reaction
The first and determining step was the implementation of the derivatization reaction, which was based on the method of the Ph.Eur. mentioned before [2].
The reaction according to Fig. 2. took place in 0.2 M NaOH/MeOH = 50/50 (v/v) mixture (later referred as solvent).
Hydrazine sulfate provided the necessary amount of hydrazine instead of the hygroscopic and unmanageable hydrazine hydrate taking into account the difference between the molecule weight of hydrazine and hydrazine sulfate. The
Conclusions
A fast and effective liquid chromatographic method was developed to determine hydrazine content in allopurinol API. For the appropriate detection, sample derivatization and an additional SPE step were necessary. The appropriate SPE sorbent was chosen and the parameters of the derivatization procedure were optimized. An HPLC method with a state-of-the-art core-shell stationary phase has been developed. The run time was cut down to 5 min. The applicability of the sample preparation and the liquid
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