Method Development, Validation, Identification and Quantification of Residual Solvents in Phenazopyridine Hydrochloride Using Head Space –Gas Chromatography Technique

Narmada Vallakeerthi1, Anisetti Ravinder Nath1, a)

1Department of Pharmacy, University College of Technology, Osmania University, Hyderabad, Telangana, India

a) Corresponding author: dranisetti2011@gmail.com, rishikaravi@gmail.com

Abstract. Good Manufacturing practices (GMP) conditions command to check the levels of residual solvents, in order to sufficiently control the quality of APIs. Organic solvents such as Methanol, Acetone, Isopropyl alcohol (IPA), Hexane, Benzene, Toluene, and chlorobenzene are often utilized in pharmaceutical industry to manufacture Active Pharmaceutical Ingredients (APIs). A selective Gas Chromatography (GC) - Head space (HS) Injection system is applied for developing and validating the method for identification and quantification of residual solvents in Phenazopyridine Hydrochloride (PPH) drug as per International conference on Harmonization (ICH) guidelines. GC with Head space sampler and a Flame ionization detector were used to monitor residual solvents in APIs. The separation was carried out on ZB-624 capillary column. As carrier gas, Nitrogen was taken in Head space method. The method is proved to be simple, accurate, precise, sensitive, robust, reliable, reproducible and linear for the Identification and quantification of Methanol, Acetone, IPA, Hexane, Benzene, Toluene, chlorobenzene in PPH drug.

1 INTRODUCTION

Residual solvents (RS), are Organic Volatile Chemicals these are used and also produced in preparing drug substances or excipients, or their products. Some solvents cause toxicity so that it should be avoided in the manufacturing of bulk drugs. The solvents that are present in the sample cannot remove completely by the practical production techniques. But selecting the solvent is considered important to synthesize drug substance that might increase the yield, or determine characteristics such as crystal form, purity, and solubility. By using solvents not having therapeutic advantage, it is needed to remove as much as required, in order to meet product descriptions, good manufacturing practices1-3. Based on ICH guideline RS categorized into three classes: Class 1 solvents: Solvents to be avoided, Class 2 solvents: Solvents to be limited, Class 3 solvents: Solvents with low toxic potential4. Gas chromatography (GC), a technique used in analytical chemistry to separate and analyze the compounds, has the capacity to vaporize compounds without spoilage. GC is employed to test the purity of a particular compound, or separate a mixture with different components. GC is also used in identifying a compound5-6. Headspace Gas Chromatography (HS-GC) is used to identify the residual solvents in pharmaceutical products, in Head space the analytes are injected Directly and this evaporates through equilibration between liquid (or solid) phase and gas phase in GC system to minimize the contamination of the GC system and prevent deterioration of GC column 7- 8. Phenazopyridine hydrochloride (PPH) is a Urinary Tract analgesic, local anesthetic that has been used in urinary tract disorders. It is an interstitial cystitis agent that exerts topical analgesic effect on urinary tract mucosa9.

Literature review reveals that there were no analytical methods for identifying and quantifying residual solvents in PPH. Therefore, the study presently develops and validates a new, simple, rapid, efficient, reproducible, quantitative method to analyze residual solvents in PPH10-13. The main aim would be to develop and validate a method for identification and quantification of residual solvents and organic volatile impurities in pharmaceutical products using HS-GC. Method development was done by HS-GC & Validation of the developed method was done by using Validation parameters.

2 EXPERIMENTAL2.1 Chemicals & Reagents

Residual solvents used for this study are Methanol (3000), Acetone (5000), IPA (5000), Hexane (290), Benzene (2), Toluene (890), Chlorobenzene (360). PPH sample gift samples obtained from Sigma labs. Methanol was purchased from Merck grade, Acetone was purchased from Fisher Scientific, IPA was purchased from Merck, Hexane was purchased from HPLC grade, Benzene was purchased from Emplura, Toluene was purchased from Emplura, Chlorobenzene was purchased from Merck, Di Methyl Sulfoxide (DMSO) Merck Life Sciences PVT ltd.

2.2 Instrumentation 2.2.1 Gas Chromatographic Conditions

The analysis is carried out for determining residual solvents by utilizing Agilent 7890 A series GC system with 7694 E head space Auto sampler Gas chromatograph & a Flame ionization detector. ZB-624 with dimensions 30 m x 0.53 mm I.D x 3µm film thickness capillary column, Stationary phase- 6% cyano propyl phenyl & 94% di methyl poly siloxane from Supelco was used for this development. The GC method for separation of residual solvents used a flow rate of 3 ml/min. Injection port temperature 225°C, and detector temperature were maintained at 250°C respectively. Linear velocity was 22.7 cm/sec. Nitrogen was used as a carrier gas. Run time: 25 min. Oven temperature was maintained at 40°C for 5 minutes, then increased to 200°C at a rate of 20°C per minute and maintained at same Temperature for about 12minutes.

2.2.2 Head space Conditions

Vial Temp: 80oC, Vial Conditioning time: 30’, Vial pressurization time: 3’, Injection volume: 1 ml Syringe Temp: 100oC, Transfer line Temp: 110oC.

2.3 Solution Preparations2.3.1 Preparation of Blank

2 ml of Dimethyl Sulphoxide (DMSO) was pipetted and transferred into a 20 ml headspace vial and sealed it immediately. The Retention time of DMSO was 12.056 minutes, and the Chromatogram of Blank was as shown in Figure 1.

2.3.2 Standard stock solution-1

10mg of Benzene was accurately weighed into a10mL volumetric flask and make up to the mark with DMSO diluent (conc: 1000 µg/ml).

2.3.2.1 Standard Stock solution-2

Weighed 75 mg of Methanol, 125 mg of Acetone, 125 mg of IPA, 7.15 mg of Hexane, 0.05 mg of Benzene Stock Solution-1, 22.36 mg of Toluene, 8.8 mg of Chlorobenzene onto a 100ml of flask and make up to the mark with DMSO. Transfer 2mL of solution into six different head space vials and sealed the vial immediately with rubber septa and metallic ring closure. Heat the sealed vial at 80oC for 30 min. The Retention times for all solvents are 4.417 for Methanol, 6.372 for Acetone, 6.599 for IPA, 7.009 for Hexane1, 7.330 for Hexane2, 7.650 for Hexane 3, 8.328 for Hexane 4, 8.967 for Benzene, 10.746 for Toluene, 11.781 for Chlorobenzene. The Chromatogram of Standard as shown in Figure 2.

2.3.3 Preparation of Sample

Transfer accurately about 0.5gm of the Phenazopyridine Hydrochloride sample into a head space vial, and add about 2 ml of DMSO to dissolve and crimp the vial. Heat the sealed vial at 80oC for 30 min. In Batch sample of PPH Methanol and Chlorobenzene were detected and the Retention times are 4.333 for Methanol and 11.715 is for Chlorobenzene. The Chromatogram of Sample was as shown in Figure 3.

2.4 Method Development

Method Development was carried out by several trials with different diluents at different chromatographic conditions, but the final trial was Optimized by using Dimethyl sulphoxide, it was selected as a diluent, chromatographic and head space conditions were mentioned in (2.2.1 and 2.2.2). Optimize all the parameters According to the acceptance limits of ICH. Blank, standard, sample each 2ml of solution was injected into the instrument and their chromatograms were recorded (figure 1, figure 2, figure 3).

FIGURE 1. Blank (Dimethyl Sulphoxide) Chromatogram.

FIGURE 2. Mixture of standard Chromatogram.

FIGURE 3. Batch Sample of Phenazopyridine Hydrochloride Chromatogram.

3 RESULTS AND DISCUSSION3.1 Method Validation

Method validation is proving the process used to confirm that an analytical procedure is employed for a particular test is suitable for its routine use. Validation parameters are System Suitability, Method precision, Specificity, LOD (Limit of Detection), LOQ (Limit of Quantification), Precision at LOQ level, linearity and Range, Accuracy (% Recovery), Ruggedness, Robustness. All the parameters are validated as per ICH guidelines.

3.1.1 System Suitability

Inject the Blank, Standard (Standard Stock solution-2) into 6 different Head space vials and crimp it and record the peak response of eluting peaks using Chromatographic and Head space conditions in order to study the system suitability parameters. chromatographic parameters like Retention time, peak area, resolution between the peaks were determined. %RSD of system suitability was below the acceptance criteria as shown in the table 1.

3.1.2 Method Precision

Method precision was carried out by injecting same sample at 100% concentration level 6 times into the instrument and %RSD values were calculated and values are below the acceptance criteria as tabulated in table1.

TABLE 1. % RSD of Standard Mixture of System Suitability, Method Precision

Solvent

%RSD System Suitability

%RSD Method Precision

Acceptance Criteria

Methanol

8.51

4.33

NMT 15%

Acetone

7.12

3.75

NMT 15%

IPA

8.72

9.61

NMT 15%

Hexane

9.03

9.39

NMT 15%

Benzene

5.92

4.42

NMT 15%

Toluene

8.89

5.59

NMT 15%

Chlorobenzene

7.97

9.30

NMT 15%

3.1.3 Specificity

Specificity study of the method was performed by injecting blank, mixed standard solution, drug sample and individual solvents such as Methanol, Acetone, IPA, Hexane, Benzene, Toluene, Chlorobenzene into the instrument and retention times were acquired from the chromatograms and %RSD was below the acceptance criteria and Retention times of individual solvents not matched with other solvents present in the mixture. The Retention times of individual solvents and mixture as shown in the table 2.

TABLE 2. Specificity of individual and Standard Mixture and % RSD.

Solvent

Retention Time in Individual Injection

Retention Time in Spiked

Injection

%RSD

Acceptance Criteria

Methanol

4.433

4.416

7.39

<10

Acetone

6.383

6.374

1.84

<10

IPA

6.610

6.598

3.32

<10

Hexane

7.014, 7.335, 7.654, 8.330

7.010, 7.330, 7.651, 8.328

5.90

<10

Benzene

8.939

8.968

3.61

<10

Toluene

10.741

10.745

1.78

<10

Chlorobenzene

11.774

11.781

2.22

<10

3.1.4 Limit of Detection (LOD) and Limit of Quantitation (LOQ), and Precision at LOQ

The detection limit and quantitation limit were determined by analyzing of a standard solution with the known concentration of residual solvents and on incorporating the minimum levels, the residual solvents detected as signal/noice ratio (S/N) was about 3.3 and quantified S/N was about 10 with an acceptable precision and accuracy as shown in the table 3.

In Precision at LOQ, Introduced 2ml of standard solution into six different head space vials and sealed the vial immediately with rubber septa and metallic ring closure. Heat the sealed vial at 80oC for 30 min. %RSD values of Precision at LOQ are below the acceptance criteria as shown in table 3.

TABLE 3. Average Signal to Noice Ratio of LOD, LOQ and %RSD of precision at LOQ solution

Solvent

LOD

LOQ

%RSD Precision at LOQ

Acceptance Criteria

Methanol

3.21

10.03

12.41

NMT 15%

Acetone

3.34

10.56

3.81

NMT 15%

IPA

3.00

10.16

9.34

NMT 15%

Hexane

3.09

9.74

10.89

NMT 15%

Benzene

2.87

9.50

8.82

NMT 15%

Toluene

3.31

10.33

3.84

NMT 15%

Chlorobenzene

3.26

10.13

8.59

NMT 15%

3.1.5 Linearity and Range

The linearity study for the method was performed by injecting 50%, 75%, 100%, 125%, 150% standard solutions in triplicate into the head space. Five point calibration curves were plotted by taking average peak areas of solvents on Y-axis and corresponding concentration on the x-axis. (Figure 4). Linearity was confirmed by keeping the statistical analysis and respective correlation coefficients and regression equations for calculating and the values are given in table 4.

A minimum of five concentration levels, including certain minimum specified ranges were determined. For assay tests, the minimum specified range was 80-120% of the target concentration. For impurity tests, the minimum range was from the reporting level of each impurity to 120% of the specification concentration range shown in table 4.

TABLE 4. correlation coefficient of Linearity data and Range

Solvent

Correlation Coefficient

Regression

Equation

Concentration (%) Range

Methanol

0.9853

y=85,18,032.9143x - 4,30,857.4286

50-150

Acetone

0.991

y = 6E+07x + 2E+06

50-150

IPA

0.9750

y=1,51,93,142.7429x - 13,24,277.7857

50-150

Hexane

0.9912

y=3,84,32,829.9429x - 1,63,812.2857

50-150

Benzene

0.9925

y =7,07,908.1143x + 25,649.5714

50-150

Toluene

0.9904

y=1,31,67,364.0571x - 1,40,613.2143

50-150

Chlorobenzene

0.9898

y=2,903,830.4571x + 1,07,544.7857

50-150

FIGURE 4. Residual solvents linearity curves

3.1.6 Accuracy

Recovery study was performed by standard addition method at three different levels of 50%, 100%, 150%. The percentage recoveries of Methanol, Acetone, IPA, Hexane, Benzene, Toluene, Chlorobenzene in the sample mixture was determined by statistical evaluation and given in table 5.

TABLE 5. Accuracy values of solvents at 50%, 100%, 150%

Solvent

Sample corrected Area (A)

Concn %

Spiked area corrected (B)

Standard area (C)

% Recovery(B-A)/C*100

%Average Recovery

Methanol

161000

50

3315451

3115472

106.42

112.84

100

6579660

5565792

118.22

150

9727345

8541306

113.89

Acetone

---

50

34443642

32450937

106.14

112.01

100

70990763

62131011

114.26

150

102878785

88976770

115.62

IPA

---

50

5176456

5207641

99.40

111.66

100

14162675

12196677

116.12

150

21183217

17733853

119.45

Hexane

---

50

13209211

16319705

80.94

94.34

100

40091368

39098590

102.54

150

40282777

40470035

99.54

Benzene

---

50

343875

426370

80.65

96.50

100

837713

797061

105.10

150

828422

798516

103.75

Toluene

---

50

8193698

7209551

113.65

115.80

100

13714165

11676702

117.45

150

25107556

21589325

116.30

Chlorobenzene

870756

50

1701311

1578060

107.81

109.92

100

2808723

2666267

105.34

150

5456389

4679321

116.61

3.1.7 Robustness and Ruggedness

Robustness study was proposed by making small but deliberate variations in parameters of the method and observing the changes. The effects of changes were ±50C in the temperature and ±2ml/min in the column flow. A Blank, standard and sample solution was injected into the instrument and calculated %RSD and shown in table 6.

Ruggedness of the method was done by injecting standard solution by changing two different analysts and different days, on two different instruments and calculate %RSD, the values are below the Acceptance Criteria NMT 15 as shown in table 6.

TABLE 6. Figures of Robustness of Flow 2.8ml/min, 3.2ml/min and Vial Temp 850c and 750c, and Ruggedness of Analyst 1 day 1 and Analyst 2 day 2 and Instrument 1,2.

Solvent

%RSD

Flow 2.8ml/min

%RSD

Flow 3.2ml/min

%RSD

Vial Temp 850c

%RSD

Vial Temp 750c

%RSD

Analyst 1

& Day 1

%RSD

Analyst 2& Day 2

%RSD

Instrument1&

Instrument 2

Methanol

2.90

3.22

6.47

3.97

8.56

4.18

4.18

Acetone

6.70

0.11

4.19

3.38

4.91

4.33

4.33

IPA

7.27

0.87

5.43

4.34

7.67

6.81

6.81

Hexane

14.32

0.19

13.11

13.60

4.73

13.57

13.57

Benzene

8.30

0.59

12.08

6.95

3.65

8.30

8.30

Toluene

7.23

0.04

3.68

3.79

7.45

7.15

7.15

Chlorobenzene

10.61

0.07

6.68

5.98

8.90

8.70

8.70

3.1.8 Batch Analysis

Batch analysis was performed by injecting a pure drug sample solution into the head space, values were shown in table 7.

TABLE 7. Results from Residual Solvent Testing in Drug Substances Using GC-HS Method

Drug Substance

Solvent Found

Solvent Found(ppm)

Weight of Sample(gm)

Acceptance Limit

Class

Phenazopyridine Hydrochloride

Methanol

82.7

0.5

3000

II

Chlorobenzene

108.8

0.5

360

II

HS-GC Method is suitable for the quantification of Residual Solvents in Phenazopyridine HCl API and can be routinely used for the analysis of samples.

CONCLUSION

From the above experimental results and parameters it was observed that this newly developed method was validated for the identification and quantification of Residual Solvents in Phenazopyridine HCl which was noticed to be simple, precise, accurate, with acceptable resolution and quick retention time and linear over the range of 50%-150%. ICH Specifications Precision and accuracy was found to be good for methanol, Acetone, IPA, Hexane, Benzene, Toluene, chlorobenzene. This method is considered to be more acceptable and cost effective and it can be effeciently applied on Identification and quantification of Residual Solvents in Phenazopyridine HCl API and can be used for the routine analysis of samples in future mainly in fields like research institutions, in industries for quality control, for approvals in laboratories.

acknowledgement

The authors Thanks the Head, Dean, principal, Department of Pharmacy, University College of Technology, Osmania University, Hyderabad, for providing lab facilities. Narmada Vallakeerthi is grateful to UGC for BSR-RFSMS (09/DOP/UCT) Fellowship under the UGC scheme 2014-15 and for financial support, I thankful to P.M.R for his Expertise and assistance throughout all aspects of study and for his help in writing the Manuscript. Thanks to Sigma labs for providing samples and providing lab facilities.

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