Validation of a stability-indicating HPLC-UV method for the quantification of acetazolamide in Oral-Mix and Oral-Mix SF

This manuscript details the modifications made to the HPLC assay method described in the USP monograph for Acetazolamide Compounded Oral Suspension.• The method was modified to allow the quantification of acetazolamide in two new suspension vehicles: Oral Mix and Oral Mix SF;• It was validated for linearity, accuracy, precision and specificity;• It was demonstrated stability-indicating and suitable for use in a stability study using these vehicles.


Method details
Overview Acetazolamide is a drug indicated for the treatment of edema, epilepsy, mountain sickness and glaucoma [1][2][3] . It is available in the USA and in Canada in various solid oral dosage forms [4 , 5] . When a liquid oral dosage form is required, it can be prepared extemporaneously according to the USP monograph for Acetazolamide Compounded Oral Suspension [6] . New liquid oral acetazolamide formulations were developed using Oral Mix and Oral Mix SF suspension vehicles and a stabilityindicating method was required in order to conduct a stability study for these new formulations. This manuscript details the modifications made to the USP methods for the quantification of acetazolamide in Oral Mix and Oral Mix SF vehicles. It also details the validation of this new method. Heptafluorobutyric acid (HFBA, lot A2716) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

Materials
Water was purified using a Milli-Q Synthesis A10 system, Millipore, Etobicoke, ON, Canada. Mobile phase A consisted of an aqueous solution of HFBA (0.15%) and mobile phase B was an acetonitrile solution of HFBA (0.15%).
All materials were used without further purification.

Sample preparation
Acetazolamide suspensions were prepared from acetazolamide bulk powder using either Oral Mix or Oral Mix SF at a concentration of 25 mg mL −1 in a mortar using a pestle.
Samples for HPLC injections were prepared by diluting the acetazolamide suspension (50 μL) using methanol (450 μL) in a 1.5-mL centrifuge tube. The mixture was vortexed (20 s) and then centrifuged (9400 g, 10 min). Supernatant (20 μL) was further diluted using mobile phase A (480 μL), vortexed (20 s) and transferred to a sealed 96-well plate. These solutions for injection had a nominal concentration of 100 μg mL −1 and were analysed immediately after preparation.

HPLC-UV conditions
The injection volume was 10 μL and all injections were conducted in duplicate. A variation of more than 0.5% between replicates triggered an investigation to identify instrumental malfunction.
The autosampler was refrigerated (5 °C) and the column oven was heated to 40 °C.  Detection wavelength and retention time were 265 nm and 3.3 min, respectively.
Limits of detection and quantification were not formally evaluated as the lower concentration of the validation range was clearly above the limit of quantification based on signal to noise ratio. This method is not intended to be used outside of its validated range.
Since robustness was not evaluated, this method requires calibration i. Furthermore, the injection of quality control samples every 20 test samples will confirm the stability of the system throughout injection runs. This stability check was applied when assessing the stability of acetazolamide oral suspensions over a period of 90 days .
HPLC response was plotted as a function of acetazolamide concentration for both Oral Mix and Oral Mix SF. A linear regression was calculated for both plots ( y -intercept was forced through zero). Coefficient of determination were respectively 0.9997 for Oral Mix and 0.9995 for Oral Mix SF.
The target concentration of test samples is 100 μg mL −1 . The range of this method is 25-150 μg mL −1 , or 25-150% of the target concentration.

Accuracy
Standard solutions of acetazolamide in mobile phase A were prepared at concentrations of 25, 50, 75, 100 and 150 μg mL −1 and analysed using the HPLC-UV method. As listed in Table 1 , in order to evaluate the effects of the matrix as well as sample preparation, the areas of the acetazolamide peaks were compared to the areas obtained during the validation of linearity and range described above. Furthermore, the concentration of acetazolamide of these samples was back calculated using the linear regression slope parameter and compared with their nominal value to establish the accuracy of the linear regression at all tested concentrations. The effect of sample preparation and matrix on the accuracy of the method resulted in recoveries ranging from 99.9% to 105.2% at all tested concentrations over the investigated range. Furthermore, the back calculated recovery varied from 97.6% to 101.7% at all tested concentrations over the method range.

Precision
Intraday precision corresponds to the coefficient of variation of the triplicated analyses performed for the validation of linearity and range detailed above. Interday variability is similarly calculated from analyses performed on three different days. These results are reported in Table 2 .
The precision of the method evaluated as the intraday CV and the interday CV were not more than 0.17% and 4.75%, respectively, at any of the tested concentrations over the method range.

Specificity
An acetazolamide suspension (25 mg mL −1 ) was prepared in Oral Mix as described above. Aliquots of this suspension (0.5 mL) were mixed with water (0.5 mL), aqueous hydrogen peroxide 3% (0.5 mL), aqueous hydrochloric acid 1 M (0.5 mL) and aqueous sodium hydroxide 1 M (0.5 mL). These four solutions were stored for 3 h at 60 °C. The acidic solution (100 μL) was neutralized using aqueous sodium hydroxide 1 M (50 μL) and diluted using methanol (350 μL). Similarly, the alkaline solution was neutralized using aqueous hydrochloric acid 1 M (50 μL) and diluted using methanol (350 μL). The water and peroxide solutions were directly diluted using methanol (400 μL). All these solutions were vortexed (20 s) and centrifuged (9400 g, 10 min). Supernatants (20 μL) were recovered and diluted in mobile phase A (480 μL) to achieve a nominal concentration of 100 μg mL −1 (prior to degradation) and analysed using the HPLC-UV method. The chromatograms obtained from these analyses were compared to the chromatograms obtained from acetazolamide suspensions in Oral Mix and Oral Mix SF (25 mg/mL) submitted to sample preparation for HPLC injection. They were also compared to the chromatograms obtained from a solution of acetazolamide in mobile phase A (100 μg/mL). Recovery values of 102%, 103%, 73% and 92% were observed following degradation in water, peroxide, alkaline and acidic conditions, respectively. As shown in Fig. 1 , no peak overlap of acetazolamide with excipients, impurities or degradation products was observed. Acetazolamide peak purity index calculated between 235 and 295 nm was not less than 0.9999 in all cases.

Conclusion
A stability-indicating method for the quantification of acetazolamide in Oral Mix and Oral Mix SF suspensions was developed and validated. This method is intended to be used for the evaluation of the stability of acetazolamide in these two oral suspension vehicles. This methods improves the USP assay method for acetazolamide compounded oral suspension. The USP method was developped and validated for these two vehicles: 1:1 mixture of Vehicle for Oral Solution, NF (regular or sugar-free), and Vehicle for Oral Suspension, NF, or Cherry Syrup, NF. The method reported in this manuscript was specifically developped and validated for acetazolamide compounded oral formulation in two other slightly different commercial vehicles: Oral Mix and Oral Mix SF. On the one hand, the latter method used a lower injection volume (10 vs. 20 μL) and a gradient elution system wich allowed sharper peaks with a better resolution. On the other hand, the USP method provided stronger signals and a simpler isocratic elution system.