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1944

Anerao et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF RELATED SUBSTANCES

METHOD BY HPLC FOR ANALYSIS OF HYDRALAZINE

HYDROCHLORIDE

Ajit Anerao*, Vikram Dighe, Ravindra Pagire, Sachin Sonavane, Nitin Pradhan

R&D centre (API), Wanbury Ltd., EL-16, TTC Industrial Estate, Mahape, Navi Mumbai

400710, India.

ABSTRACT

Process related impurities associated with the synthesis of Hydralazine

hydrochloride active pharmaceutical ingredient (API) was detected by

high performance liquid chromatography (HPLC). The impurities are

synthesized in laboratory and structure elucidation is done by mass

spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR)

and infra-red spectroscopy (FTIR). Gradient HPLC method is

developed for quantitation on Inertsil C18 column and validated for

parameters such as specificity, accuracy, precision, linearity,

robustness and ruggedness. The LOD and LOQ of all specified

impurities are also determined.

KEYWORDS: Hydralazine hydrochloride, impurity characterization,

method development and validation, HPLC, FTIR, mass and NMR.

INTRODUCTION

Hydralazine hydrochloride USP is an antihypertensive drug, available as 10, 25, 50 and 100

mg tablets for oral administration. Its chemical name is 1-hydrazinophthalazine

monohydrochloride. Therapy is initiated gradually increasing dosages. Start with 10 mg four

times daily for the first 2-4 days, increase to 25 mg four times daily for the balance of the first

week. For the second and subsequent weeks, increase dosage to 50 mg four times daily. In a

few resistant patients, up to 300 mg daily may be required for a significant antihypertensive

effect.[1]

Hydralazine is a direct-acting smooth muscle relaxant used to treat hypertension by

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 5.210

Volume 4, Issue 11, 1944-1965 Research Article ISSN 2278 – 4357

Article Received on

17 Sep 2015,

Revised on 06 Oct 2015,

Accepted on 27 Oct 2015

*Correspondence for

Author

Ajit Anerao

R&D centre (API),

Wanbury Ltd., EL-16,

TTC Industrial Estate,

Mahape, Navi Mumbai

400710, India.


1945


acting as a vasodilator primarily in arteries and arterioles. By relaxing vascular smooth

muscle, vasodilators act to decrease peripheral resistance, thereby lowering blood pressure.

The analytical methods on impurity detection and identification are reported in some

literatures. Hydralazine hydrochloride is a Pharmacopoeia product.[2]

Synthetically prepared

hydralazine hydrochloride was injected on high performance liquid chromatography (HPLC)

method from USP, a late eluting unknown impurity peak at retention time 129 minutes in

chromatogram was observed during analysis. An unknown process impurity at 129 minutes

with a broad peak was observed. Peak width was 9.4 minutes. It is difficult to quantify the

impurity with such a broad peak in accuracy point of view. During literature search this

impurity is found reported.[3]

Separate method reported in the same literature by using UPLC

where only this impurity is quantified. We have developed new method of analysis where the

unknown impurity (impurity-F) can be quantified along with another four impurities

mentioned in the pharmacopeia by HPLC. In USP impurity-A, B, C and E are reported.

During process development studies, impurities were detected in both crude and pure samples

of Hydralazine hydrochloride using a newly developed gradient reversed phase HPLC

method. One more unknown impurity (impurity-D) was observed consistently during process

optimization at RRT 5.9. This unknown impurity was not reported in any of the synthetic

process related to Hydralazine hydrochloride active pharmaceutical ingredient (API).

The impurity profile of the drug substance is critical for its safety assessment and

manufacturing process. It is mandatory to identify and characterize the impurities in

pharmaceutical product, if present above the accepted limit of 0.10%.[4]

In this present article

complete characterization of both unknown impurities (impurity D and impurity F) was done

using NMR, mass and IR. HPLC method is developed for quantitation of both unknown

impurities as well as another four specified impurities as per United State Pharmacopoeia

(USP).[2]

However, so far there is no published report, describing the complete

characterization and simultaneous quantitation of this unknown process related impurity in

Hydralazine hydrochloride API. This article also describes the analytical method validation

as per ICH, International conference on harmonization, validation of analytical procedures[5]

by using HPLC for quantitative determination of specified and unspecified impurities.


1946


MATERIAL AND METHOD

1.0 Instrumentation and liquid chromatographic conditions

HPLC method was performed using a Shimadzu-2010 CHT HPLC system with UV detector

and Lab Solution software. Separation was achieved with the mixture of mobile phase-A:

buffer and mobile phase-B: methanol in gradient elution with timed programme Tmin/A: B:

T0/90:10; T10/90:10; T45/35:65; T50/30:70; T60/30:70; T61/90:10 and T70/90:10 with flow rate

1.0 mL/min. The column temperature was maintained at 30°C and sample cooler temperature

is 10°C. Ultraviolet detection was performed at 230nm. Injection volume is 10μL and run

time is 70.0 minutes. HPLC column is Inertsil ODS-3V, 250mm length, 4.6mm internal

diameter and 5µm particle size.

Table-1 Reagents and chemicals used in the experimentation.

Sr. No Reagents/Solvents Grade Make

1 Methanol HPLC Merck or its equivalent.

2 Orthophosphoric Acid HPLC Merck or its equivalent.

3 Potassium dihydrogen phosphate AR Merck or its equivalent.

4 Water HPLC Siemens Lobostar or its equivalent.

Table-2 Standards used in the experimentation.

Sr. No Standard Batch No. Potency Manufacturer

1 Hydralazine hydrochloride M01216 99.9% USP

2 Impurity-A: Phthalazine SP-002-188 99.7% Simson Pharma

3 Impurity-B: 2-Formyl

benzoic acid SP-MKBF3349V 99.2% Simson Pharma

4 Impurity-C: 1-Phthalazinone SP-01518JJV 99.7% Simson Pharma

5

Impurity-D: 1-[(2)-2-

phthalazin-1(2H)-ylidene

hydrazino] phthalazine

HLZ/38/Imp/II/253/09 95.2% Wanbury Ltd.

6 Impurity-E: 1-

Chlorophthalazine SP-10164459 90.3% Simson Pharma

7 Impurity-F: 1, 1’-Hydrazine-

1, 2-diyl-dipthalazine HLZ/38/dimer/224/32A 94.5% Wanbury Ltd.

1.1 Preparation of Buffer

Weigh and dissolve 0.34 g of potassium hydrogen phosphate in 1000 mL of water. Adjust pH

2.5 with dilute ortho-phosphoric acid, mix well and filter though 0.45µ filter.

1.2 Preparation of standard and sample solutions

Diluent: Adjust pH of water to 3.2 with dilute ortho phosphoric acid.


1947


1.2.1 Standard stock solution

Weigh and transfer 10 mg each of hydralazine hydrochloride USPRS, impurity-D and

impurity-F and 15 mg each of impurity-A, impurity-B, impurity-C and impurity-E reference

standard into 100 mL volumetric flask. Add 50 mL of solvent mixture (i.e. mixture of

acetonitrile and methanol 1:1 v/v) and sonicate to dissolve. Dilute up to the mark with diluent

and mix.

1.2.2 Standard solution-A

Transfer 5.0 mL of standard stock solution in to 50 mL volumetric flask and dilute up to the

mark with diluent.

1.2.3 Standard solution-B

Further dilute 5.0 mL of standard solution-A in to 50 mL volumetric flask and dilute up to the

mark with diluent.

1.2.4 System suitability solution

Weigh and transfer 50 mg of hydralazine hydrochloride USPRS in to 50 mL volumetric flask.

Add diluent and sonicate to dissolve. Transfer 5.0 mL of standard solution-A and dilute up to

the mark with diluent and mix.

1.2.5 Test solution

Weigh and transfer 50 mg of hydralazine hydrochloride sample in to 50 mL volumetric flask.

Add diluent and sonicate to dissolve. Dilute up to the mark with diluent and mix.

1.2.6 Hydrochloric acid solution

Weigh and transfer 44.3 ml of concentrated hydrochloric acid (35%) in to 100 mL volumetric

flask. Dilute up to the mark with diluent and mix (18.3% hydrochloric acid with respect to

test concentration of hydralazine hydrochloride).

Note

1) Standard stock solution and test solution to be prepared fresh and inject immediately

during analysis.

2) The peaks due to hydrochloric acid to be identified from hydrochloric acid solution at

retention time 2.0 minute, 2.2 minute and 2.5 minute to be disregarded in test solution.


1948


1.3 Procedure

Inject blank (diluent), system suitability solution, Standard solution-B, Hydrochloric acid

solution and Test solution in the chromatograph. The retention time of hydralazine peak is

about 5.5 minutes under given chromatographic condition (figure 1 to 4).

Table-3 Relative retention times of specified impurities.

Compound Relative retention time (RRT)

Hydralazine hydrochloride 1.0

Impurity-A: Phthalazine 2.4

Impurity-B: 2-Formyl benzoic acid 4.5

Impurity-C: 1-Phthalazinone 5.0

Impurity-D: 1-[(2)-2-phthalazin-1(2H)-ylidene

hydrazino] phthalazine 5.9

Impurity-E: 1-Chlorophthalazine 6.5

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 7.5

1.4 Evaluation of system suitability

The system is suitable for analysis, if and only if,

1) The relative standard deviation (RSD) of peak area of hydralazine, impurity-A,

impurity-B, impurity-C, impurity-D, impurity-E and impurity-F in six replicate injections of

standard solution-B should not be more than 5.0%.

2) Resolution between the peak of impurity-B and impurity-C of system suitability

solution should not be less than 5.0.

3) Tailing factor of the hydralazine peak of system suitability solution should not be

more than 2.8.

1.5 Calculation

Disregard the peaks due to blank and hydrochloric acid. Calculate known impurities and

unknown impurities by following formula.

% specified impurity= Area of specified impurity peak in Test solution x Conc of specified

impurity in std. solution-B (mg/mL) x P1 x 100

Avg. area of specified impurity std. solution-B x Conc of test solution (mg/mL) x 100.

% any unspecified impurity= Area of any unspecified Imp. in Test solution x Conc of

Hydralazine hydrochloride in std. solution-B (mg/mL) x P2 x 100

Avg. Area of Hydralazine peak in std. solution-Bx Conc. of test solution (mg/mL) x 100.


1949


% Total unspecified impurities = Sum of area of all unspecified Imp. in Test solution x Conc.

of Hydralazine hydrochloride in std. solution-B (mg/mL) x P2 x 100

Average area of Hydralazine peak in std. solution-Bx Conc. of sample solution (mg/mL) x

100.

Where, P1=Potency of specified impurity reference standard

P2=Potency of Hydralazine hydrochloride reference standard

Total Impurities = Σ of total specified impurities and total unspecified impurities.

Figure-1: Blank chromatogram.

Figure-2: System suitability chromatogram-All impurities spiked at 100% level.

5.3 mins-Hydralazine, 13.3 mins-impurity A, 25.6 mins-impurity B, 28.4 mins-impurity C,

32.9 mins-impurity D, 36.6 mins-impurity E and 42.0 mins-impurity F.


1950


Figure-3: Standard chromatogram.

Figure-4: Test solution chromatogram.

2.0 Mass spectroscopy

Mass of the isolated impurities was performed on Waters Micro Mass (MS) DI mass lcm

instrument.

3.0 NMR spectroscopy

1H NMR measurement of the isolated impurities was performed on Bruckner NMR with 400

MHz instrument. Number of protons was reported on the δ scale (ppm) relative to DMSO.

4.0 FTIR spectroscopy

The FTIR spectrum of isolated impurity was recorded in the solid state as KBr powder

dispersion using (Perkin-Elmer, Beaconsfield, UK) spectrum one FT-IR spectrometer.

5.0 HPLC METHOD VALIDATION

Method validation is closely related to method development. When a new method is being

developed, some parameters are already being evaluated during the “development stage,”

while in fact, this forms part of the “validation stage.”


1951


5.1 Specificity and force degradation

The ability of the method to determine accurately and specifically the analyte of interest in

the presence of other components in a sample matrix (that may be expected to be present in

the sample matrix) under the stated conditions of the test (specificity = 100% selectivity).

Specificity of the method was evidenced by comparing blank, hydralazine, impurity-A,

impurity-B, impurity-C, impurity-D, impurity-E and impurity-F. As well as all impurities are

spiked into hydralazine hydrochloride test solution [figure-2].

From the experimental data, there are no interfering peaks at the retention times of

hydralazine and specified impurities are observed from the chromatogram. All specified

impurities are well resolved without any interference is observed from the spiked

chromatogram.

Table-4 Specificity: Resolution and peak purity.

Compound Relative retention time (RRT) Resolution Peak purity

Hydralazine hydrochloride 1.0 1.00 Passing

Impurity-A: Phthalazine 2.1 9.8 Passing

Impurity-B: 2-Formyl benzoic acid 3.9 21.2 Passing

Impurity-C: 1-Phthalazinone 4.3 8.4 Passing


hydrazino] phthalazine 5.1 14.9 Passing

Impurity-E: 1-Chlorophthalazine 5.6 8.2 Passing

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 6.5 16.6 Passing

Force degradation study is performed by exposing the sample to heat at 105⁰C for 24 hours,

ultra-violate light for 48 hours, sample treated with base 0.5 N sodium hydroxide for 1 hour

and with acid 5N HCl for 24 hours. Hydralazine hydrochloride is found stable when exposed

to heat and UV light. In basic condition it is unstable and forming impurity-B about 27.0%.

In acidic condition one unknown impurity is found increased at RRT 4.8 about 0.16% after

24hrs exposure to 5N hydrochloric acid. All degradant impurities are well resolved and peak

purity of all peaks is passing.

5.2 Solution stability

Drug stability in Active Pharmaceutical Ingredient is a function of storage conditions and

chemical properties of the drug and its impurities. Conditions used in stability experiments

should reflect situations likely to be encountered during actual sample handling and analysis.

Stability data are required to show that the concentration and purity of analyte in the sample


1952


at the time of analysis corresponds to the concentration and purity of analyte at the time of

sampling.

The solution stability was checked from HPLC peak area of standard solution-B. The

standard solution-B was kept at room temperature for 18 hours. Considerable change in the

peak area of hydralazine, impurity-D, impurity-E and impurity-F has been observed.

Table-5 Solution stability: Peak area at room temperature of initial and after 18 hours.

Compound Initial peak area After eighteen hours at room

temperature peak area

Hydralazine 25197 40930

Impurity-A: Phthalazine 221150 220135

Impurity-B: 2-Formyl benzoic acid 49364 53622

Impurity-C: 1-Phthalazinone 96890 119927


hydrazino] phthalazine 35243 1665

Impurity-E: 1-Chlorophthalazine 39827 15822

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 35454 23762

As observed in above study the standard solution-B is not stable at room temperature so the

same solution was prepared freshly and analysed and it was kept in sample cooler at

temperature 10⁰C for 15 hours. The solution stability was checked from HPLC peak area of

standard solution-B. It was observed that there was no considerable change in peak area of

hydralazine as well as all specified impurities at sample cooler temperature 10⁰C.

Table-6 Solution stability: Peak area at sample cooler 10⁰C of initial and after 15 hours.

Compound Initial peak area After fifteen hours at

10⁰C peak area

Hydralazine 31439 30592

Impurity-A: Phthalazine 208949 212420

Impurity-B: 2-Formyl benzoic acid 56273 53696

Impurity-C: 1-Phthalazinone 90549 94596


hydrazino] phthalazine 37444 34155

Impurity-E: 1-Chlorophthalazine 40499 38060

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 34683 32724

The solution stability was ascertained from HPLC peak area of test solution. The test solution

was kept in sample cooler temperature of 10⁰C for 6 hours; it was observed that there was no

considerable change in peak area and area percent of hydralazine peaks as well as other

impurities are also not changed.


1953


Table-7 Solution stability: Peak area of hydralazine at sample cooler 10⁰C of initial and

after 15 hours in test solution.

Hydralazine peak area Hydralazine peak area %

Initial 33859013 99.96%

After six hours 33572244 99.95%

5.3 Linearity and response factor

The ability of the method to obtain test results proportional to the concentration of the analyte

within a given range. It was evaluated by linear regression analysis, which was calculated by

the least square regression method. Response factor is also found out by calculating slope of

the hydralazine hydrochloride and is compared with the related impurity.

Under the experimental conditions, the peak area vs. concentration plot for the proposed

method was found to be linear over the range of 25% to 150% of the specified limit with a

correlation coefficient as tabulated below.

Table-8 Linearity: Correlation coefficient and RRF.

Compound Correlation

coefficient (R2)

Relative response factor with respect

to hydralazine hydrochloride

Hydralazine hydrochloride 0.9996 1.00

Impurity-A: Phthalazine 0.9999 4.70

Impurity-B: 2-Formyl benzoic acid 0.9997 1.18

Impurity-C: 1-Phthalazinone 0.9999 2.08


hydrazino] phthalazine 0.9998 1.12

Impurity-E: 1-Chlorophthalazine 0.9996 0.89

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 0.9972 1.06

5.4 Limit of detection

The limit of detection (LOD) is the point at which a measured value is longer than the

uncertainty associated with it. It is the lowest concentration of analyte in a sample that can be

detected but not necessary quantified.

LOD = 3.3 × SO / b

Where, SO and b are standard deviations and slope of the calibration line, respectively.

5.5 Limit of quantitation

The limit of quantitation is the lowest concentration or amount of analyte that can be

determined quantitatively within an acceptable level of repeatability precision and trueness.

Limit of quantitation (LOQ) = 10.0 × SO/b


1954


Table-9 Limit of detection and quantification

Compound Limit of detection (%) wrt

test concentration

Limit of quantification (%)

wrt test concentration






hydrazino] phthalazine 0.003 0.010


Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 0.017 0.051

5.6 Accuracy

Accuracy can be defined as the closeness of agreement between a test result and the accepted

reference value. Accuracy of the method was determined by recovery study.

Analytical method may be considered validated in terms of accuracy if the mean value is

within ±20% of the actual value. Recovery of specified impurities was found in the range of

80.0% to 120.0%, which was well within the acceptance criteria.

Table-10 Accuracy: Recovery of specified impurities.

Compound Recovery at

25% level

Recovery at

5o% level

Recovery at

100% level

Recovery at

150% level

Impurity-A: Phthalazine 99.2 96.2 96.4 96.2

Impurity-B: 2-Formyl benzoic acid 80.7 82.0 89.8 86.3

Impurity-C: 1-Phthalazinone 102.9 98.4 97.9 96.6


hydrazino] phthalazine 101.6 98.3 98.3 96.4

Impurity-E: 1-Chlorophthalazine 97.1 93.6 93.8 89.6

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 109.9 112.2 114.6 116.6

5.7 Ruggedness

The (intra-laboratory tested) behaviour of an analytical process when small changes in

environment and/or operating condition are made.

The ruggedness of the method was evaluated by estimating % RSD of standard solution-B

and system suitability by two different analysts using different HPLC instrument and

columns on different days. Results are tabulated below.


1955


Table-11 Ruggedness: Details of ruggedness experiment.

Ruggedness parameters Analyst 1 Analyst 2

Date of analysis 22nd

May 2015 23rd

May 2015

HPLC instrument No. ADL/HLC/07 ADL/HLC/05

HPLC column serial No. IB7150961 IB7144500

Table-12 Ruggedness: System suitability comparison.

Compound % RSD Tailing factor of

hydralazine peak

Resolution between

impurity-B and impurity-C

Analyst 1 Analyst 2 Analyst 1 Analyst 2 Analyst 1 Analyst 2


2.45 2.60 9.09 9.39




Impurity-D: 1-[(2)-2-phthalazin-

1(2H)-ylidene hydrazino]

phthalazine

1.00 0.67


Impurity-F: 1, 1’-Hydrazine-1, 2-

diyl-dipthalazine 1.57 0.48

Table-13 Ruggedness: Batch analysis result comparison

Batch No.: HLZ/38/IV/253/04

Compound Analyst 1 Analyst 2

Impurity-A: Phthalazine Below detection limit Below detection limit

Impurity-B: 2-Formyl benzoic acid Below detection limit Below detection limit

Impurity-C: 1-Phthalazinone Below quantification limit Below quantification limit


hydrazino] phthalazine 0.03% 0.02%

Impurity-E: 1-Chlorophthalazine Below detection limit Below detection limit

Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine 0.04% 0.05%

Any unspecified impurity 0.03% 0.03%

Total impurities 0.14% 0.14%

5.8 Robustness

Robustness is a measure of the capacity of the analytical procedure to remain unaffected by

small but deliberate variations in method–performance parameters, which provides an

indication of its reliability during normal usage.

Robustness of the method was determined by analyzing the system suitability solution with

deliberate change in the parameters like (a) flow rate of mobile phase ± 0.1 ml/min, (b)

column temperature ± 5°C and (c) mobile phase pH ± 0.2.


1956


5.8.1 Change in flow rate

It was observed that at a flow rate of 0.9 ml/min and 1.1 mL/minute, resolution between

impurity-B and impurity-C peak of system suitability solution were found 8.81 and 9.49,

respectively. The system suitability parameters were within desired limits.

Table-14 Robustness: Effect of change in flow rate.

Flow rate Tailing factor of

hydralazine peak

Resolution between impurity-

B and impurity-C

0.9 mL/minute 2.55 8.81

1.0 mL/minute 2.49 9.07

1.1 mL/minute 2.42 9.49

5.8.2 Change in column temperature

It was observed that resolution between impurity-B and impurity-C in system suitability

solution is 8.77 and 9.53 when column temperature changed from 25°C to 35°C respectively.

The system suitability parameters were within desired limits.

Table-15 Robustness: Effect of change in column temperature.

HPLC column

temperature

Tailing factor of

hydralazine peak

Resolution between

impurity-B and impurity-C

25⁰C 2.58 8.77

30⁰C 2.49 9.07

35⁰C 2.37 9.53

5.8.3 Mobile phase pH

It was observed that resolution between impurity-B and impurity-C in system suitability

solution is 9.16 and 9.26 when the pH of the buffer is changed from 2.3 to 2.7 but tailing

factor is increased at pH 2.3.

Table-16 Robustness: Effect of change in mobile phase pH.

Mobile phase pH Tailing factor of

hydralazine peak

Resolution between impurity-

B and impurity-C

2.3 2.73 9.16

2.5 2.49 9.07

2.7 2.35 9.26

6.0 Impurity Formation/Synthesis and Characterization

Impurity-A, Impurity-B, Impurity-C and Impurity-E are reported in the USP monograph.


1957


N

N

NHNH2

.HCl

Figure-5: Structure of Hydralazine hydrochloride.

Figure-6: Structure of Impurity-A. Figure-7: Structure of Impurity-B.

Figure-8: Structure of Impurity-C. Figure-9: Structure of Impurity-E.

Impurity-D and impurity-E are synthesized and characterized in-house as described below.

6.1 Impurity-D: 1-[(2)-2-phthalazin-1(2H)-ylidene hydrazino] phthalazine

N

N

NHN

NH

N

Figure-10: Structure of impurity-D.

Impurity-D, 1-[(2)-2-phthalazin-1(2H)-ylidene hydrazino] phthalazine, is formed during the

synthesis of Hydralazine Hydrochloride, wherein 1-chloro phthalazine is treated with 80%

hydrazine hydrate in Isopropyl alcohol at 0-100C and then heated to 60-65

0C for 2-3 hrs.

Progress of the reaction is monitored on HPLC. Reaction mass filtered at 60-650C. Unload

solid product and dried at 40-450C.

Impurity-D, i.e. 1-[(2)-2-phthalazin-1(2H)-ylidene hydrazino] phthalazine can also be

synthesized as follows:

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1958


Phthalzinone is treated with 1-phthalazinyl hydrazine (Hydralazine free base) in Isopropyl

alcohol (IPA) at 25-300C and then heated to 60-65

0C for 7-8 hrs. Progress of the reaction is

monitored on HPLC. Reaction mass filtered at 60-650C. Unload solid product and dried at

40-450C.

Molecular weight: 288.30

Molecular formula: C16H12N6

6.1.1 Mass spectrum

Molecular weight of the parent compound: 288.3

Base peak observed at Mass: 289.3 (M+1) + ve mode (figure-11)

Table-17 Impurity-D: Proton NMR interpretation (figure-12 and 13).

Table-18 Impurity-D: IR interpretation (figure-14).

Frequency cm-1

Functional groups / interpretation

3404 & 3331 -N-H stretch amine

3050 -C-H stretch aromatic

1606 -C=N aromatic stretch

1556-1606 -C=C stretch aromatic (in ring)

1382-1476 -C-C stretch aromatic (in ring)

1329 -C-N stretch

Figure-11: Mass spectrum of impurity-D.

Chemical Shift Observed (ppm) Details Protons

7.56-7.65 6H;multiplate Aromatic proton (a,b,c)

7.86 2H;multiplate Aromatic proton (d)

8.64-8.67 2H;multiplate Aromatic proton (e)

11.6 2H;singlate N-H protons (f)

Total 12-Protons


1959


Figure-12: Proton NMR of impurity-D.

Figure-13: D2O exchange NMR of impurity-D.

Figure-14: IR spectrum of impurity-D.


1960


6.2 Impurity-F: 1, 1’-Hydrazine-1, 2-diyl-dipthalazine

N

N

NHNH

N

N

Figure-15: Structure of impurity-F.

Impurity-F, i.e. 1, 1’-Hydrazine-1, 2-diyl-dipthalazine which is also called as Dimer impurity

can be synthesized as follows.

Chloro Phthalazine is treated with 1-phthalazinyl hydrazine (Hydralazine free base) using

Isopropyl alcohol (IPA) as solvent at 60-650C for 4-8 hrs. Progress of the reaction is

monitored on TLC/HPLC. After completion of reaction cool the reaction mass to 25-300C

under stirring, further cool the reaction mass to 10-15 and maintain for 1.0 hr, filtered the

solid and washed with isopropyl alcohol. Wet solid obtained is again stirred with DM water

for 1.0 hr at 25-300C. Filter and unload the solid product and dried at 40-45

0C.

Molecular weight: 288.30

Molecular formula: C16H12N6

6.2.1 Mass spectrum

Molecular weight of the parent compound: - 288.3

Base peak observed at Mass: - 289.1. (M+1) + ve mode (figure-16)

Table-19 Impurity-F: Proton NMR interpretation (figure-17 and 18).

Chemical Shift Observed (ppm) Details Protons

7.79-8.28 6H;multiplate Aromatic proton (a,b,c)

8.90-8.92 2H;multiplate Aromatic proton (d)

9.10-9.11 2H;multiplate Aromatic proton (e)

14.8 2H;singlate N-H protons (f)

Total 12-Protons


1961


Table-20 Impurity-F: IR interpretation (figure-19).

Frequency cm-1

Functional groups / interpretation

3410 -N-H stretch amine

2912 -C-H stretch aliphatic

1556-1606 -C=C aromatic stretch

1432-1505 -C-C stretch aromatic (in ring)

1317-1362 -C-N stretch

Figure-16: Mass spectrum of impurity-F.

Figure-17: Proton NMR of impurity-F.


1962


Figure-18: D2O exchange NMR of impurity-F.

Figure-19: IR spectrum of impurity-F.

RESULTS AND DISCUSSIONS

As per existing method of USP 41(2) In-process revision one of the process impurity was

eluting at 129 minutes. The impurity peak was not sharp and symmetrical so it was difficult

to quantify that impurity at 0.10% level. In the proposed method of analysis the same

impurity-F is eluting at retention time 42 minutes (RRT 7.7). The peak is sharp and

symmetrical and can be quantified at low level about 0.10%. All four impurities listed in USP

as well as another two process impurities are well resolved in the proposed method.


1963


During solution stability study it was observed that the specified impurities are getting

degraded at room temperature. So the impurity solution and test solution was kept at 10⁰C

and it was observed that hydralazine hydrochloride and related impurities are stable in

solution form at 10⁰C. On basis of solution stability study sample cooler with temperature

10⁰C is recommended.

Specificity and selectivity of the method is confirmed by injecting all specified impurities

individually and by performing force degradation study. Hydralazine hydrochloride is found

stable in solid form when exposed to heat and UV light. In basic and acidic condition it is

unstable and forming impurity-B and an unknown impurity at RRT 4.8 respectively. All

degradant impurities are well resolved and peak purity of all peaks is passing.

Accuracy of the method is proved by performing recovery study where known quantities of

the specified impurities are spiked in to the sample. Recovery of all specified impurities is

observed within 80.0% and 120.0%. The results are found consistent in deliberately made

changes in the different instruments, different lot of columns on different days which proves

that the method is rugged and robust.

Future development

During experimentation it is observed that the impurity-B that is 2-Formyl benzoic acid is not

stable. It converts in to another impurity which is eluting at the retention time 35 minutes. But

it is not interfering with other specified or unspecified impurities. One more Impurity-E that

is 1-Chlorophthalazine is not stable at room temperature as well as at 2 to 8⁰C in solid form.

Due to stability of both impurity standards fresh preparation of the standard stock solution is

recommended. To avoid or overcome the above mentioned practical difficulties response

factors of all impurities are determined and can be implemented instead of injecting and

calculating impurities against their respective standards.

CONCLUSION

The proposed method does not require any laborious clean up procedure before measurement.

The validation data demonstrate good accuracy, which proves the reliability of the proposed

method. Hence, the validated method can be used for routine determination of related

substances in hydralazine hydrochloride in quality control laboratories in the pharmaceutical

industry.


1964


ACKNOWLEDGEMENT

Thanks to Mr. P. V. Pashupathy, President (API), Wanbury Limited for his constant support

and the management of the Wanbury Limited.

REFERENCES

1. James E F Reynold, Kathleen Barfitt Martindale; The extra pharmacopoeia 31st edition;

Published by the council of royal pharmaceutical society of Great Britain, 1996; 885.

2. United State Pharmacopoeia 41(2) In-Process Revision: Hydralazine Hydrochloride,

2015.

3. Manohar V. Lokhande, Nitin G. Rathod, Mukesh Kumar Gupta; Structural elucidation,

Identification, quantization of process related impurity in Hydralazine Hydrochloride

HR/AM- LC MS/MS, NMR and FTIR technique; IOSR Journal of Applied Chemistry,

Nov. – Dec. 2013; 6(2): 05-15.

4. ICH, International conference on harmonization; Impurities in New Drug Substances,

2006; Q3A(R2).

5. [5] ICH, International conference on harmonization; validation of analytical procedures;

text and methodology, 2005; Q2(R1).

6. European Pharmacopoeia 8.0: Hydralazine Hydrochloride, 2437-2438.

7. G. Santhosh1*, G. Nagasowjanya1, A. Ajitha, Dr. V. Uma Maheshwara Rao; Analytical

method development and validation of simultaneous estimation of isosorbide and

hydralazine hydrochloride in tablet dosage form by RP-HPLC; International Journal of

Pharmaceutical Research & Analysis, 2014; 4(5): 307-311.

8. Ronald J. Molles Jr., Yves Garceau; Quantitation of hydralazine hydrochloride in

pharmaceutical dosage forms by high performance liquid chromatography; Journal of

chromatography A, 1985; 347: 414-418.

9. Tibor Urbanyi, Arthur O'Connell; Simultaneous automated determination of hydralazine

hydrochloride, hydrochlorothiazide, and reserpine in single tablet formulations;

Analytical Chemistry, 1972; 44(3): 565–570.

10. K. Siddappa*, Mallikarjun Metre, Tukaram Reddy, Mahesh Tambe; Simple And

Selective Spectrophotometric Methods For The Determination Of Hydralazine

Hydrochloride In Bulk Drug And In Pharmaceutical Formulations Based On The

Oxidation-Reduction Reactions; Analytical Chemistry, 7(8): 652-658.

11. World Health Organization; IARC monographs on the evaluation of the carcinogenic risk

of chemicals to humans, 1980; 24: 85-100.

http://pubs.acs.org/action/doSearch?ContribStored=Urbanyi%2C+Tibor

http://pubs.acs.org/action/doSearch?ContribStored=O%27Connell%2C+Arthur


1965


12. Leela Madhuri Pola1* and Gowri Sankar D2; A novel validated stability indicating RP-

LC method for simultaneous quantitative estimation of hydralazine hydrochloride and

isosorbide dinitrate in bulk drug and combined tablet dosage form; World Journal of

Pharmacy and Pharmaceutical Sciences, 4(02): 1154-1169.

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