Hydralazine

Abstract

The objective of this study was to evaluate the chemical stability of hydralazine hydrochloride 0.1% and 1% when prepared extemporaneously from commercially available hydralazine bulk powder. The oral solutions were prepared to contain preservatives, sweeteners, a humectant and diluting agents. They were prepared flavored and nonflavored and stored in duplicate glass and plastic bottles. Each of the flavored preparations was stored at 5 deg C, 25 deg C/60% relative humidity and 40 deg C/75% relative humidity. Samples from the bottles of concentrations stored under these conditions were taken for a period of up to 92 days. Each of the nonflavored preparations was stored at 5 deg C. Samples from the bottles stored under these conditions were taken for a period of up to 72 days. Samples were taken from glass and plastic bottles on the designated sampling days for both flavored and nonflavored preparations, and the samples were analyzed for drug concentration using a stability-indicating high-pressure liquid chromatography analytical technique. Degradation occurred faster in the 0.1% oral solution than in the 1% solution at all the studied conditions. At 40 deg C, a considerable amount of drug loss was observed for both the 0.1% and the 1% oral solutions for the flavored preparations. For both flavored and nonflavored preparations, the lowest studied temperature condition (5 deg C) appeared to be the most favorable for the preparations, with 93% or more of the initial potency retained by day 29.

Introduction

Hydralazine hydrochloride is found to be stable as a crystalline solid.1 It is also found to be stable in a distilled-water solution for weeks at room temperature.1 Hydralazine in solution decomposes at pH values greater than 7 to form phthalazine. This rate of decomposition is dependent on the concentration of anion, pH and temperature. However, at higher temperatures of 275 to 280 deg C, crystalline hydralazine hydrochloride degrades to form hydrazine. Hydrazine has been found to be carcinogenic in humans.2 Therefore, to eliminate the presence of hydrazine, extra caution is necessary in the compounding preparation of hydralazine hydrochloride solutions.

Previous studies of compounded solutions of hydralazine hydrochloride have identified stability problems of hydralazine when in the presence of some excipients used in the preparation.3-9 Gupta et al investigated the stability of hydralazine in the presence of various sweetening agents and observed instability, finding hydralazine to be stable in 0.28 M mannitol at 24 deg C after 21 days.3 Other investigators4-9 have evaluated the compatibility of hydralazine with other compounds, such as edetate sodium and sodium bisulfite.

The purpose of this study was to determine the stability of hydralazine hydrochloride with or without raspberry flavor in aqueous solutions containing sorbitol solution, methylparaben, propylparaben, propylene glycol and aspartame. Each preparation with flavor was assayed for drug concentration, and apparent pH was measured for days 0, 2, 6, 9, 14, 22, 29, 51, 69 and 92 at studied temperatures of samples stored at 5, 25 and 40 deg C. Each preparation without flavor was assayed for drug concentration; and apparent pH was measured for days 0, 7, 14, 22, 30, 43, 60 and 72 at the temperature of 5 deg C only.

The stability of hydralazine hydrochloride was studied in solutions containing hydralazine hydrochloride at 0.1% and 1% concentrations, which represent the concentrations usually prepared by pharmacists engaged in compounding practices.

The results of the study with flavored solutions were published in Pharmacopeial Forum.10 The genesis behind preparation of oral solutions without flavor was based on inconclusive evidence on the reaction of flavors with hydralazine to form hydrazone derivatives. The results of the studies with flavored and nonflavored solutions are discussed below.

Materials and Methods Chemicals

Chemicals used for this study were hydralazine hydrochloride (Lot FD132, Spectrum Pharmacy Product, Inc., Tucson, Arizona), sorbitol solution 70% 40 g (Lot 950514, Gallipot, Inc., St. Paul, Minnesota), methylparaben 65 mg (Lot 950396, Gallipot, Inc.), propylparaben 35 mg (Lot 940765, Gallipot, Inc.), propylene glycol (Lot 941273, Gallipot, Inc.), aspartame (Lot T405040, Nutrasweet, Chicago, Illinois), high-performance liquid chromatographic-grade (HPLC) methanol (Lot B 1937, Burdick & Jackson, Muskegan, Michigan), glacial acetic acid (Lot 656019, Fisher Scientific Co., Fairlawn, New Jersey), Milli-Q Purified Water USP and rapberry flavor (Gallipot, Inc.).

Equipment

The following equipment was used for this study: a Hewlett Packard 1050 series autosampler and detector with Hewlett Packard Chem Station software (Avondale, Pennsylvania), a (mu)Bondapak phenyl 30 cm x 3.9 mm ID standard column (Model E5136C, Waters Corporation, Milford, Massachusetts), and an Orion pH meter model 720 A (Orion Research, Inc., Beverly, Massachusetts), which was used for apparent pH measurements at room temperature and was calibrated using pH 4 and pH 7 buffered solutions.

Preparation of Formulations

Sample preparations for stability studies were prepared by three pharmacists. The formulas were prepared according to Table I in two different concentrations (0.1% and 1%) with and without raspberry flavor. All blank and active-drug solutions were prepared in duplicate and stored in 4-oz amber polyethylene terephthalate plastic bottles and 4-oz amber glass bottles. Preparations with flavor were stored at 5 deg C, 25 deg C/60% relative humidity (RH) and 40 deg C/75% RH; and preparations without flavor were stored at 5 deg C. Each solution was visually assessed for color and clarity before it was poured into the bottle. Approximately 2 mL of solution from each of the bottles, which were labeled 0.1 % Solution, 1% Solution and Blank Solution, was removed for analysis and apparent pH measurement. Flavor-containing preparations stored at 5, 25 and 40*C were sampled at 0, 2, 6, 9, 14, 22, 29, 51, 69 and 92 days. Preparations without flavor stored at 5 deg C were sampled at 0, 7, 14, 22, 30, 43, 60 and 72 days. All samples were tested for pH and analyzed for drug concentration.

Blank Solutions and Standard Solutions

Blank solutions were prepared in the same manner as described in Table 1 but without the use of hydralazine hydrochloride. Flavored and nonflavored solutions were prepared, and were labeled as Blank Solution With Flavor, and Blank Solution Without Flavor, respectively. Standard solutions were prepared using Hydralazine Hydrochloride USP reference standard to yield the desired diluting range for standard-curve preparations and controls. The mobile-phase solution was used as the diluting solvent, and the desired concentrations ranged from 10 (mu)g/mL to 100 (mu)g/mL.

Method of Analysis

To establish retention times, a reverse-phase HPLC analytical technique was used to analyze hydralazine. The validation study was performed according to procedures provided in the United States Pharmacopeia (USP) 25AVational Formulary 20, general chapter "Validation of Compendial Methods." Validation included linearity, precision, specificity, accuracy limit of quantification and column effect (Okeke CC, Valid,tion Report, 1995).11,12 The formulation for the 0.1% and 1% hydralazine hydrochloride solutions is shown in Table 1.

Chromatographic Conditions

The mobile phase contained 2% aqueous acetic acid and methanol (70:30 v/v). The mobile phase was used as the diluting solvent for the preparation of standard and simple solutions. The flow rate was 1.0 mL/minute, the detector wavelength was 295 nm, the injection volume was 10 (mu)L and the temperature was ambient. The run time was 10 to 15 minutes.

The 1% hydralazine hydrochloride solution was diluted 10 times to yield the same final concentration as the 0.1% hydralazine hydrochloride solution. Drug concentrations were determined using a liquid chromatograph. Each of the solutions was injected twice. The relative standard deviation for replicate injections of each of the sample or standard solution was found to be less than 1.0%. The tailing factor for the analyte peak was found to range from 0.9 to 1.0. The r^sup 2^ value for the standard curve of hydralazine hydrochloride solution ranged from 0.9989 to 0.9999.

The retention time of hydralazine was 5.352 (x 0.053) minutes. Samples were analyzed using an autosampler programmed for single injections. For the study with flavor, aliquot stability samples of hydralazine hydrochloride oral solutions (0.1% and 1%) were diluted to yield the desired final concentration (20 (mu)g/mL) for analysis. A hydralazine hydrochloride standard solution of 20 ((mu)g/mL was assayed periodically as a control (See Figure 1 for the representative chromatogram.). The peak-area response from injections of standard hydralazine hydrochloride solutions with concentrations of 10, 20, 40, 60, 80 and 100 (mu)g/mL was used for the standard curves produced by linear regression of peak areas against hydralazine hydrochloride concentrations. The standard curve was linear with r^sup 2^ values of 0.9995 to 1.000 over the working range of hydralazine concentrations. The intraday and interday coefficients of variation were less than 1%.

Antimicrobial Testing

Only the preparation without flavor was studied for microbial presence. The study was performed by Gibraltar Labs, Inc. Samples for both 0.1% and 1% solutions were prepared without flavors to determine whether a product or formulation prevented multiplication or killed high levels of inoculated organisms or whether a preservative-free material was intrinsically hostile to microbial growth or maintenance of viability.

Each sample was divided into 5 aliquots of 20 mL and transferred into sterile 25-mm test tubes. Each test system was derived as appropriate from either trypticase soy agar (TSA) or Sabouraud's dextrose agar (SDA). Each sample was inoculated with 0.1 nK of each inoculum suspension to achieve approximately 105 to 106 cfu/mL and was mixed thoroughly with a vortex mixer. Verification of the bacterial inoculum was made by aerobic plate count on TSA. Verification of mold and yeast inoculum was made by aerobic plate count on SDA.

The inoculated samples were incubated in sealed vessels at 20 to 25 deg C, and recovery of viable organisms was performed at the cited intervals by standard plate count in TSA of SDA supplemented with 4% Tween 20 and 0.5% lecithin. Verification of the neutralization of preservatives was accomplished by streaking low numbers of challenge organisms onto lowdilution plates. Recovery plates were incubated at 30 to 35 deg C (48 hours) for bacteria and at 20 to 25 deg C (5 to 7 days) for filamentous mold and yeast.

Data Simulation

The data collected were computer analyzed to obtain the apparent first-order rate constants, and predicted values were determined by the use of regression analysis to determine the best plot. A graph of log concentration was plotted against days to get the apparent first-order rate constants. The independent variable X (time in days) was regressed against the dependent variable Y (ie, percent remaining of hydralazine hydrochloride) to yield a trend line equation (Y = a X + b) with slope (a) and Y intercept (b) values. The predicted values of the dependent variable, percent remaining of hydralazine hydrochloride, were part of the output of the regression analysis.

Results and Discussion Assay

The assay was shown to be stability indicating after studies of hydralazine under stressful chemical and physical conditions. Samples were subjected to extreme heat (72 deg C), pH (0.1 N hydrochloric acid, 0.1 N sodium hydroxide base) and oxidation stress (peroxide 3%). Addition of a strong base resuited in at least 60% degradation with eluting degradation peaks. Heat and acid stressing yielded at least 5% and peroxide at least 30% degradation (see Figure 2 for representative chromatogram). No interfering peaks were observed during the study period (Okeke CC, Project Report, 1996).10

Stability Results

The behavior of the hydralazine hydrochloride solutions 0.1% and 1 % with flavor, stored in both glass and plastic bottles at 25 and 40 deg C, is summarized in Tables 2 and 3. As expected, the data show that hydralazine hydrochloride in solution degraded faster at 40 deg C than at 25'C. The data for the hydralazine hydrochloride solution 1 % stored in glass and plastic bottles indicated similar degradation tendencies at 25 and 40 deg C, but the hydralazine hydrochloride solution 0.1% data showed that degradation was faster in plastic bottles than in glass bottles. Further examination of the data showed that, in both glass and plastic bottles on day 22, the 0.1% solution was over 90% degraded at 40*C and was approximately 10% degraded at 25 deg C in both glass and plastic bottles. For the 1% solution in glass and plastic bottles at 25 and 40 deg C, the degradation was less than 10%.

To allow for prediction of degradation tendencies of hydralazine hydrochloride, plots were made of the percentage that remained as a function of time. The lines that have the r2 values are shown in Tables 2 and 3. These values ranged from 0.68 to 0.97, which indicated that a strong tendency to linearity did not exist, possibly due to a complex reaction.

The results shown in Tables 2 and 3 that were obtained with the 0.1% and 1% solutions indicated that storage at 25 and 40 deg C would not be satisfactory and that a complicated reaction made prediction of stability unreliable.

The studies were extended to PC in order to determine whether this temperature would provide satisfactory conditions for storage of both 0.1% and 1% hydralazine hydrochloride solutions. In addition, since the flavor may have provided undesirable opportunities for chemical reaction, stability studies in the absence of flavor were included.

Stability Observations

All results of the stability studies of the 0.1% and 1% solutions with and without flavor and stored in glass and plastic bottles at PC are reported in Tables 4 and 5. The following observations have been summarized from these data.

* The 0.1% solution stored in glass bottles, and the 1% solution stored in both glass and plastic bottles, were within +/- 10% of the initial value. The 0.1% solution in plastic bottles indicated an approximate loss of 12%, which exceeded this limit.

* The level of decomposition was less for the 0.1% solution without flavor and stored in plastic bottles than for the 0.1 % solution stored in glass bottles.

* The level of decomposition was less for the 1% solution without flavor and stored in glass bottles than for the 1 solution stored in plastic bottles.

* The level of decomposition was less for the 0.1% and the 1% solutions with flavor and stored in glass bottles than for the 0.1% and the 1% solutions stored in plastic bottles.

* For the 0.1 % and the 1% solutions, logarithmic plots of the percentage hydralazine hydrochloride remaining versus time yielded straight lines with excellent r^sup 2^ values (Tables 4 and 5). The various plots are shown in Figures 3 through 10.

* The linear plots cited in item 5 (above) produced equations that allowed calculation of predicted values of the percentage remaining of hydralazine hydrochloride.

* The slopes of the linear plots are shown in Figures 1 through 8 and in Table 6 and are presented as first-order degradation rate constants. However, it is recognized that when the extent of decomposition is less than 20%, the distinction between reaction rate orders is not important. Therefore, no argument is presented in this study about the order of the hydralazine reaction. The data do, however, adhere to the requirements of a first-order reaction.

* At 5 deg C, the predicted drug stability is always better in solution at 92 days when there is no flavor in this product (Tables 2 and 3).

The predicted values were in close proximity with actual data. Similar observations were discussed for solutions with flavor in referenced articles (Okeke CC, Validation Report, 1995).10

Other work that has discussed hydralazine degradation at 25 and 40 deg C has observed that at 25 deg C/60% RH, the 0.1% solution showed a 10% loss of hydralazine on day 22, and the 1% solution remained within 10% of the labeled amount up to day 92 (Okeke, CC, Validation Report, 1995).10

Appearance of the Solution

Change in color due to drug loss was always more pronounced in the 1% solution than in the 0.1% solution. The observed change in color was usually from slightly off-white to clear yellow.

Apparent pH

For the solutions with and without flavor stored at 5 deg C, the observed apparent pH range was 4.15 to 4.29 (+/- 0.14) for the 0.1% solution stored in glass bottles and was 3.98 to 4.18 (+/- 0.2) for solution stored in plastic bottles during the study. Similarly, the observed pH range for the 1% solution stored in glass bottles was 4.09 to 4.18 (+/- 0.09) and 3.98 to 4.13 (+/- 0.15) for solution stored in plastic bottles. The observed difference in apparent pH was less than 5% for any of the solutions.

The pH decreased from 4.27 to 3.86 and further to 3.32 in 51 days for the 0.1% solution stored at 25 deg C/60% RH in either glass or plastic bottles. For the 1% solution, the pH changed from 3.97 to 3.47. The apparent pH changes at 25 deg C were much larger than the apparent pH changes at 5 deg C.

Antimicrobial Testing

Both solutions of hydralazine hydrochloride (0.1% and 1%) in solutions without flavor and stored in glass or plastic bottles conformed to the requirements of USP 23 and 25 against all organisms tested and were within the limits specified for antimicrobial testing. Visually, no microbial growth was detected for any of the samples.

Summary

This study indicates that storage at 5 deg C affords excellent protection for both 0.1% and 1% hydralazine hydrochloride solutions without flavor, if a 30-day beyond-use date is to be recommended. There were changes in the apparent pH values for both the 0.1% solution and the 1% solution when stored at 25 deg C (0.4 to 0.5 pH units); however, for both solutions at 5 deg C, the change was 0.2 pH units or less. It is not possible to

interpret these changes since it was not the intention of this study to examine the mechanism of hydralazine hydrochloride decomposition. There were no visible signs of microbial growth in any sample.

The color change from slightly off-white to clear yellow was more pronounced in the 1% oral solution than in the 0.1% solution. There were no detectable changes in odor for either solution.

The bioavailability of hydralazine in this formulation has not been evaluated. However, for typical oral dosage forms (tablets), food may enhance the bioavailability by reducing first-pass metabolism in the gastrointestinal wall.13 Consistent administration in relation to meals is recommended.13 To mask the bitter taste of the drug, palatability can be improved by mixing the solution with fruit juice or applesauce immediately prior to administration. This procedure is recommended in pediatric or geriatric dosing.

Conclusion

As is evident from the preceding discussion, the storage of hydralazine hydrochloride solution preparations at 5 deg C ensures a shelf life of up to 30 days compared with a higher temperature with or without flavor added. Any reaction between hydralazine and raspberry flavor could not be clearly determined in this study. Therefore, the results of this study lead us to recommend not using flavor during extemporaneous preparation. To ensure palatability, however, we recommend that patients mix the hydralazine hydrochloride solution with fruit juice or applesauce immediately prior to administration.

References

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9. Gyllenhaal 0, Gronberg L, Vessman J. Determination of hydrazine in hydralazine by capillary gas chromatography with nitrogen-selective detection after benzaldehyde derivatization. J Chromatogr 1990;511:303315.

10. Nairn JG. Proposed monographs for hydralazine hydrochloride oral solutions 0.1% and 1.0%. Pharmacopeial Forum 1997;23:4931-4933.

11. United States Pharmacopeial Convention, Inc. The Ninth Supplement to United States Pharmacopeia 23/National Formulary 18. Rockville, MD: US Pharmacopeial Convention, Inc.; 1999:4556.

12. United States Pharmacopeial Convention, Inc. United States Pharmacopeia 25/National Formulary 20. Rockville, MD:US Pharmacopeial Convention, Inc.; 2002:2256-2258.

13. United States Pharmacopeial Convention, Inc. United States Pharmacopeia DI 18. Rockville, MD:US Pharmacopeial Convention, Inc.; 1998;1:1598.

Claudia C. Okeke, PhD

Thomas Medwick, PhD

Graham Nairn, PhD

Sheila Khuspe, BSc

Lee T. Grady, PhD

United States Pharmacopeial Convention, Inc. Rockville, Maryland

Address correspondence to: Claudia C. Okeke PhD, United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD 20852. E-mail: cco@usp.org

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