Detection and Evaluation of process related impurities in Obeticholic Acid Drug material using HPLC Method

 

Kishore Gaddam¹*, Srinivas Kumbam¹, Trivikram Reddy Gundala¹,

Surendranath Reddy Reddiwary¹, Gangi Reddy Nallagondu Chinna²*

¹Analytical Development, Hetero R&D, TSIE, Balanagar, Hyderabad, India.

²Department of Chemistry, Yogi Vemana University, Kadapa, India.

 

ABSTRACT:

Obeticholic acid (OBE) is being used to treat primary biliary cirrhosis and cholangitis. An HPLC with refractive index detection method for eight process related substances of OBE was developed and verified for use in quality assurance laboratories for regular analysis. The separation and analysis were performed on YMC Triart C18 (3.0 µm particle size, 250 mm × 4.6 mm) column. Mobile phase employed consisted of 0.01N potassium phosphate buffer (3.0 ± 0.05 pH, set with 0.1% orthophosphoric acid) and acetonitrile at 45:55 (v/v) ratio. The method established for determination of eight impurities in OBE was validated and verified in keeping with International Council for Harmonisation guidelines. This method can be used for routine analysis of eight impurities in OBE bulk samples.

 

 

INTRODUCTION:

Pharmaceutical impurities are unnecessary molecules that remain as contaminant in the active pharma ingredients or/and drug formulation product. Impurities in active pharma ingredients or/and drug formulation product may occur during production or may be derived from precursor materials, reagents, intermediates, solvents, or may be by-product of reactions^1,2. Impurities also may occur during drug formulation product development as a consequence of the drug substance’s characteristic instability or inconsistency with the excipients added, or through interactions with packaging components^3,4. The quantities of different impurities observed in active pharma ingredients or/and drug formulation product will ascertain the ultimate safeness of the pharmaceutical end product⁵. The detection, quantification and control of impurities are therefore a very crucial part during the process of developing drugs. Techniques like GC-MS^6,7, LC-MS^8-12, HPLC^13-15, UPLC¹⁶ and TLC¹⁷ were used commonly to detect, quantify and control impurities.

 

Different regulatory agencies like “International Conference on Harmonization”, “United States Food and Drug Administration”, and “European Medicines Agency” have incorporated specification limits which restrict impurity concentration levels existing in active pharma ingredients and drug formulation product^18-21. The detection, quantification and control of impurities all throughout the development of active pharma ingredients or drug formulation product makes it easier to identify the potential risk correlated with the toxic effects of any drug when taken by patients²².

 

Primary biliary cirrhosis is an chronic condition that often tends to lead to liver damage resulting in liver failure that necessitates liver transplantation in the end stages²³. Obeticholic acid (OBE) is synthetically revised bile acid with effective farnesoid X nuclear receptor agonist activity. OBE is being used to treat hepatic sicknesses including primary biliary cirrhosis and cholangitis²⁴.

 

The eight process related impurities of OBE are HPA[(4R)-4((3R,5S,8R,9S,10S,13R,14S)-3-Hydroxy10,13-dimethyl-7-oxohexadecahydro-1H-cyclo penta[α]phenanthren-17-yl)pentanoic acid], EDE [3α,7α-Dihydroxy-6-ethyliden-5β-cholan-24-oic acid], EUC [3α,7β-Dihydroxy-6α-ethyl-5β-cholan-24-oic acid], CDC [(R)-4-((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-Dihydroxy-10,13-dimethylhexadeca hydro-1H-cyclo penta[a] phenantren-17-yl) pentanoic acid], EUD [3α,7β -Dihydroxy-6β-ethyl-5β-cholan-24-oic acid], BDA [3α-Hydroxy-6-ethylidin-7-oxo-5β-cholan-24-oic acid], EHP [(R)-4-((3R,5S,6R,8S,9S,10S,13R,14S,17R)-6-Ethyl-3-hydroxy-10,13-dimethyl-7-oxohexadecahydro-1H-cyclo penta[a]phenanthren-17-yl) pentanoic acid], ECD [3α,7α-Dihydroxy-6β-ethyl-5β-cholan-24-oic acid]. The structures of OBE and impurities are provided in Figure 1. The specification limits for HPA, EUC, EUD, BDA, EHP, ECD were found as 0.10% concentration while for EDE and CDC were 0.15% concentration based on ICH Q3A guideline of new drug substances¹⁸.

 

Only one HPLC method coupled with UV and mass spectrometry (electrospray ionization) detection reported by Douša et al., was seen in literature for the quantification of OBE and its 18 related compounds²⁵. For the first time ever, here we present an HPLC method coupled with refractive index detector to quantify 8 impurities (HPA, EDE, EUC, CDC, EUD, BDA, EHP, ECD) simultaneously in bulk drug of OBE.

 

MATERIALS AND METHODS:

IMPURITIES, OBE, CHEMICALS AND SOLVENTS:

HPA (98.3% purity), EDE (87.0% purity), EUC (92.9% purity), CDC (89.9% purity), EUD (91.9% purity), BDA (82.8% purity), EHP (71.1% purity) and ECD (93.9% purity) reference impurity samples were procured from Hetero R & D (Telangana, India). The reference bulk sample of OBE was also procured from Hetero R & D (Telangana, India). Analytical grade potassium dihydrogen phosphate (Merck, India) and orthophosphoric acid (Merck, India), HPLC grade acetonitrile (Merck, India) and Milli Q water (Milli Q system) were employed in this study.

 

Figure 1: Structures of OBE and eight impurities

 

INSTRUMENTATION AND CONDITIONS:

The chromatographic separation, detection and quantification of HPA, EDE, EUC, CDC, EUD, BDA, EHP, ECD simultaneously in bulk drug of OBE was performed on Waters HPLC system (model number e2695) coupled with refractive index detector (model number 2414) employing YMC Triart C18 (3.0 µm particle size, 250 mm × 4.6 mm) column. Mobile phase employed consisted of 0.01N potassium phosphate buffer (3.0 ± 0.05 pH, set with 0.1% orthophosphoric acid) and acetonitrile at 45:55 (v/v) ratio. The mobile phase mixture was filtered as well as degassed with a 0.45μ membrane filter prior to use. The same mobile phase mixture was employed as diluent. The optimized values of other parameters include: isocratic method of elution, 0.7 ml/min flow rate, detection of impurities was done using refractive index with detector sensitivity of 64, 50 ^oC of column temperature, 50 ^oC of detector temperature and 20 µl of injection volume. The total of 60 min time was needed for single analysis.

 

SOLUTIONS OF IMPURITIES, REFERENCE OBE AND OBE TEST SAMPLES:

Impurities stock standards (HPA, EDE, EUC, CDC, EUD, BDA, EHP, ECD) were prepared separately in mobile phase at a concentration of 0.2 mg/ml. Standard mixture solution containing the eight impurities at a concentration of 0.10% (HPA, EUC, EUD, BDA, EHP and ECD) and 0.15% (EDE and CDC) in mobile phase was produced by diluting aptly the impurities stock standards. Further dilutions of impurities stock standards were carried out to obtain mixed impurities solutions with in target range concentrations of 0.028% - 0.150% (HPA), 0.055% - 0.226% (EDE), 0.058% - 0.225% (CDC), 0.040% - 0.151% (EUC), 0.037% - 0.150% (EUD), 0.045% - 0.150% (BDA), 0.027% - 0.150% (EHP) and 0.042% - 0.150% (ECD). Reference solution of standard OBE was prepared at a concentration of 0.02 mg/ml. To prepare OBE test sample, 200 mg OBE was balanced and dissolved in 10 ml of mobile phase (20 mg/ml concentration), sonicated and filtered.

 

GENERAL PROCEDURE TO EVALUATE THE EIGHT IMPURITIES IN OBE TEST SAMPLE:

The column was equilibrated for a minimum time period of 60 min. Infused the diluent solution (n=1) into the system and the chromatogram was recorded. Programmed the data processor to inhibit the peaks due to blank solution. Infused the reference OBE solution (n=2) into the system and documented the chromatograms. Separately injected OBE test solutions into the system and recorded the chromatograms. The peak area counts of impurities in test sample and peak count of OBE in reference solutions were determined. The percentage of impurities in the test sample were calculated using the equation below.

 

Where, AT - Impurity peak area response in test solution; AS - OBE peak area response in reference solution; CS - OBE concentration (mg/ml) in reference solution; CT - Test solution concentration (mg/ml); and P - Potency (%w/w) of OBE standard.

 

Figure 2: Chromatogram of eight impurities and OBE sample

 

RESULTS AND DISCUSSION:

The aim of this exercise was to establish a sensible and effective HPLC with refractive index detection system to evaluate eight impurities in OBE. Owing to their identical structures (Figure 1) the separation of OBE and its eight impurities is critical. As part of the development work, various stationary phases (Zodiac C18, Inertsil C8, Inno C18, YMC Triart C18 and Luna phenyl hexyl) with distinct particle sizes (3.0 μm and 5.0 μm) were used. Different mobile phases, like diverse proportions of phosphoric acid buffer: acetonitrile, potassium phosphate buffer: acetonitrile and phosphoric acid buffer: acetonitrile: methanol solutions, were examined. Better peak shape and peak separation (Figure 2) of eight impurities were observed on the YMC Triart C18 (3.0 µm particle size, 250 mm × 4.6 mm) column with 50^oC temperature. Impurities were enumerated in OBE using 0.01N potassium phosphate buffer (3.0 ± 0.05 pH, set with 0.1% orthophosphoric acid) and acetonitrile at 45:55 (v/v) ratio as the mobile phase with 0.7 ml/min flow rate.

 

VALIDATION:

The process mentioned was validated in alignment with criteria of the International Council for Harmonization (ICH)¹⁹.

 

SYSTEM SUITABILITY:

OBE reference solution was used to evaluate system suitability. The OBE reference solution was infused into the system and chromatograms were documented. The theoretical plates number and OBE tailing factor were determined. The values of theoretical plates and tailing factor were 39684 and 1.05, respectively. The values of column efficiency and tailing factor for the OBE were satisfactory as the theoretical plates number was significantly higher than 3000 plates per meter and the tailing factor was less than 2.

 

SPECIFICITY:

Blank diluent, standard impurities mixture solution (concentration 0.10% each of HPA, EUC, EUD, BDA, EHP and ECD; and 0.15% each of EDE and CDC) and OBE sample solution added with eight impurities at same concentration standard impurities mixture solution were used to evaluate specificity. These solutions have been infused into the system and the respective chromatograms are included in Figure 3. Interference was not detected due to blank diluent at the retention times of impurities. For each impurity, the relative retention time and elution order obtained from the standard impurities mixture solution and impurities spiked OBE sample solution were matched (Table 1).

 

Figure 3: Chromatogram of [a] Diluent blank [b] Standard impurities mixture solution [c] Impurities spiked OBE sample

 

Table 1: Variation between relative retention times of impurities

Impurity	Relative retention time
	Standard solution	Spiked OBE solution	Variation
HPA	0.34	0.33	0.01
EDE	0.45	0.43	0.02
CDC	0.46	0.44	0.02
EUC	0.47	0.45	0.02
EUD	0.54	0.52	0.02
BDA	0.57	0.55	0.02
EHP	0.59	0.57	0.02
ECD	0.83	0.79	0.04
OBE	1.00	1.00	0.00

 

DETECTION AND QUANTIFICATION LIMITS:

Detection and quantification limits were calculated by using the response deviation and slope of linearity curve as suggested by ICH. The detection limit values were HPA - 0.009%, EDE - 0.018%, EUC - 0.013%, CDC - 0.019, EUD - 0.012%, BDA - 0.015%, EHP - 0.009%, ECD - 0.014%. The quantification limit values were HPA - 0.028%, EDE - 0.055%, EUC - 0.040%, CDC - 0.058%, EUD - 0.037%, BDA - 0.045%, EHP - 0.027% and ECD - 0.042%. Percent RSD for area counts of each impurity from six injections of quantification limit solution was determined to confirm precision at the quantification limit level and the values ranged from 1.07% to 4.93%. Percent recovery of each impurity from three injections of quantification limit solution was determined to confirm accuracy at the quantification limit level and the values ranged from 97.2% to 106.8%.

 

LINEARITY:

Linearity from the quantification limit concentration to 150% of the specification limit concentration for each impurity was verified. Linear regression information analysis was used to determine the intercept, slope and correlation coefficient (Table 2). In the percent concentration range evaluated, a value of > 0.99 for correlation coefficient presented that the method is linear.

 

Table 2: Linearity information of impurities

Impurity	Linearity (%)	RSD (%)*	Trend line equation	Correlation coefficient (r2)
HPA	0.028 - 0.150	0.15 - 2.05	A= 904755 c + 937	0.9993
EDE	0.055 - 0.226	0.52 - 4.55	A = 674790 c - 576	0.9981
CDC	0.058 - 0.225	0.95 - 5.10	A = 717350 c - 938	0.9984
EUC	0.040 - 0.151	0.09 - 4.66	A = 793313 c - 831	0.9976
EUD	0.037 - 0.150	0.33 - 2.06	A = 716126 c - 235	0.9997
BDA	0.045 - 0.150	0.53 - 1.95	A = 681411 c - 186	0.9982
EHP	0.027 - 0.150	0.55 - 3.21	A= 698615 c + 160	0.9985
ECD	0.042 – 0.150	0.30 - 3.12	A= 690036 c + 109	0.9993

*Range of relative standard deviation for three peak area counts

A = Peak area counts                                               x = Percentage concentration of impurity

 

Table 3: Precision information of the system and method

System precision
Drug	Peak area count*	RSD (%)
OBE	72087	4.53
Method precision
Impurity	% Content determined**	RSD (%)
HPA	0.110	0.55
EDE	0.191	0.42
CDC	0.180	0.28
EUC	0.137	1.31
EUD	0.110	0.73
BDA	0.103	3.69
EHP	0.108	2.69
ECD	0.113	0.44

*average for six peak area counts

** average for six determinations

 

Table 4: Accuracy information of the method

Impurity	50% specification level		Specification level		150% specification level
	Content added (%)	Content recovered (%)*	Content added (%)	Content recovered (%)*	Content added (%)	Content recovered (%)*
HPA	0.0501	109.3	0.1003	109.8	0.1504	107.1
EDE	0.0752	104.9	0.1505	102.3	0.2257	109.3
CDC	0.0751	100.7	0.1502	97.5	0.2253	101.2
EUC	0.0502	102.2	0.1005	103.2	0.1507	106.0
EUD	0.0501	106.0	0.1003	110.0	0.1504	104.0
BDA	0.0501	107.2	0.1001	104.1	0.1502	100.6
EHP	0.0500	108.1	0.1000	106.8	0.1499	106.2
ECD	0.0501	111.4	0.1002	112.3	0.1503	110.8

* average for three determinations

Table 5: Ruggedness information of the method

Impurity	Condition 1		Condition 2		Overall results
	Content determined (%)*	RSD (%)	Content determined (%)*	RSD (%)	Content determined (%)*	RSD (%)
HPA	0.110	0.55	0.103	1.17	0.107	2.26
EDE	0.191	0.42	0.196	0.56	0.194	1.34
CDC	0.180	0.28	0.179	1.68	0.180	1.17
EUC	0.137	1.31	0.138	0.72	0.137	1.09
EUD	0.110	0.73	0.105	0.76	0.107	2.90
BDA	0.103	3.69	0.101	1.19	0.102	2.84
EHP	0.108	2.69	0.107	1.31	0.110	2.00
ECD	0.113	0.44	0.108	1.48	0.110	2.55

* average for six determinations

Condition 1: Day 1, column 1, instrument 1 and analyst 1

Condition 2: Day 2, column 2, instrument 2 and analyst 2

 

Table 6: Robustness information of the method

Impurity	Flow rate 0.6 ml/min		Flow rate 0.8 ml/min		pH value 2.8 units		pH value 3.2 units
	Content determined (%)*	RSD (%)	Content determined (%)*	RSD (%)	Content determined (%)*	RSD (%)	Content determined (%)*	RSD (%)
HPA	0.097	0.62	0.100	0.60	0.101	1.68	0.102	3.53
EDE	0.176	1.31	0.185	0.32	0.186	1.24	0.194	1.96
CDC	0.157	0.64	0.169	1.78	0.169	0.89	0.171	1.46
EUC	0.122	3.28	0.128	1.80	0.132	1.59	0.135	0.89
EUD	0.100	2.00	0.097	1.55	0.102	3.14	0.108	0.93
BDA	0.089	2.92	0.091	3.85	0.096	1.25	0.093	3.76
EHP	0.098	1.73	0.100	0.60	0.101	2.48	0.101	2.87
ECD	0.107	1.96	0.104	0.96	0.100	0.00	0.104	1.15

* average for three determinations

 

PRECISION:

The percentage of RSD for OBE peak area from six replicate injections of OBE reference solution was determined for system precision evaluation. Table 3 summarized the values of system precision. The percentage of RSD for eight impurities content from six replicate injections of OBE sample solutions added with eight impurities (concentration 0.10% each of HPA, EUC, EUD, BDA, EHP and ECD; and 0.15% each of EDE and CDC) was determined for method precision evaluation. Table 3 summarized the values of method precision. The precision has been proven by percentage of RSD results which are within limits in both system and method precision investigations.

 

ACCURACY:

The accuracy was tested by injecting 3 OBE sample solutions spiked at three separate concentration levels of each impurity studied (50% of specification limit, specification limit, and 150% of specification limit). Table 4 summarized the values of method accuracy. The accuracy has been proven by percentage of recovery results which are within limits.

 

STABILITY OF OBE TEST SOLUTION:

The OBE sample solution added with eight impurities at their specification concentration limit was stored at room temperature. Using developed method, evaluated the solution at different time intervals (0 hr, 12hr, 24 hr and 48 hr). Solution was regarded stable for 48 hours as until this time no significant variation in percentage content of impurities has been observed.

 

RUGGEDNESS:

Ruggedness was explored using OBE sample solution added with eight impurities at the concentration limit of their specifications. Using developed method, the solution was evaluated with two separate columns, two different analysts on two dissimilar days, and the percentage of RSD was computed Table 5. The ruggedness has been proven by percentage of RSD results which are within limits

 

ROBUSTNESS:

Robustness checks were carried out on the OBE sample solution added with eight impurities at the concentration limit of their specifications in order to experimentally check whether small intentional changes in flow rate (± 0.1 ml/min change) and pH value (± 0.2 units change) will affects the effectiveness of the established method. Table 6 summarized the values of method robustness. The robust has been proven by percentage of RSD results which are within limits.

 

CONCLUSION:

In the work described herein, HPLC technique was effectively applied for the development of an analytical method for the simultaneous determination of eight selected process related impurities in OBE. This method is capable of efficient separation and determination of all eight impurities in the OBE. The method established was verified for system suitability, precision, robustness, ruggedness, sensitivity, linearity and accuracy. It has been demonstrated to be suitable for the expected purpose.

 

ACKNOWLEDGEMENTS:

The authors thank Hetero R&D, TSIE, Balanagar, Hyderabad-18 for providing required facilities to carry out this research work.

 

CONFLICTS OF INTEREST:

The authors declare that they do not have conflicts of interest.

 

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Accepted on 22.11.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(11):5618-5624.