RP-HPLC Method Development and Validation of Upadacitnib (UDB) and Tofacitinib (TFC) in Bulk and pharmaceutial Dosage Form.

Analytical chemistry is inherently a quantitative Science. Whether determining the concentration of a species in a solution, evaluating equilibrium constant, measuring a reaction rate or drawing a correlation between a compounds structure and its reactivity. Analytical chemists make measurements and perform calculations.  

SPECTROPHOTOMETRIC METHOD :- 

This is most accurate method for determining the concentration of substance in solution, but the instruments are, of necessity, more expensive. A Spectrophotometer may be regarded as refined filter photoelectric photometer which permits the use of continuously variable and more nearly monochromatic bands of light. 

A DOUBLE BEAM UV - VISIBLE ABSORPTION SPECTROMETER 

Most modern general purpose UV - Visible Spectrophotometers are double-beam instruments which cover the range between 200 - 800 nm by a continuous automatic scanning process producing the spectrum as a pen trace on calibr. 

 DRUGPROFILE  

Fig:Structure of UDB

Molecular formula :C17H19F3N6O  

Solubility :Rapidly soluble inWater and Ethanol.  

TFC is an oral Janus kinase(JAK) inhibitor indicated for the treatment of autoimmune diseases such as rheumatoid arthritis, psoriaticarthritis, and ulcerative colitis.The molecular structure of TFC is shown in Fig  

Fig:  Structure of TCF

Molecular formula :C16H20N6O  

Molecular weight : 312.37 gm / mole. Characteristics: Bitter, crystalline and odourless.   

Solubility: Rapidly soluble in Water and Ethanol.  

METHODOLOGY  

PART A: DERIVATIVE SPECTROSCOPY  

METHOD A: FIRST ORDER DERIVATIVE SPECTROSCOPY  

Preparation of stock solutions:  

Standard UDB 100 mg and TFC 100 mg were each weighed and transferred to separate 100 ml volumetric flasks and dissolved in distilled water. The flasks were shaken and the volumes were made up to the mark with distilled water to give solutions containing 1000 µg/ml of UDB and 1000µg/ml of TFC, respectively. From each of these stock solutions,10ml was pipette out and placed into separate 100 ml volumetric flasks, and the volumes were made up to the mark with distilled water to give solutions containing 100 µg/ml of UDB and 100 µg/ml of TFC, respectively.  

Selection of analytical concentration ranges:  

From the standard stock solutions of UDB and TFC, appropriate aliquots were pipetted out into separate10 ml volumetric flasks ,and dilutions were made with distilled water to obtain working standard solutions of concentrations ranging from 10 to 150 µg/ml. The absorbance of the UDB solutions was measured at 267 nm, while the absorbance of the TFC solutions was measured at 287 nm. For both UDB and TFC, the analytical concentration range for the standard solutions was found to be 10-100 µg/ml. T  

METHOD B: SECOND ORDER DERIVATIVE METHOD  

Preparation of stock solutions:  

Standard UDB 100 mg and TFC 100 mg were each weighed and transferred to separate 100 ml volumetric flasks and dissolved in distilled water. The flasks were shaken, and the volumes were made up to the mark with distilled water to give solutions containing 1000 µg/ml of UDB and 1000µg/ml of TFC, respectively. From each of these stock solutions,10ml was pipette out and placed into separate 50 ml volumetric flasks. The volumes were made up to the mark with  

Distilled water to give solutions containing 200µg/ml of UDB and 200µg/ml of TFC, respectively.  

Selection of analytical concentration ranges:  

From the standard stock solutions of UDB and TFC, appropriate aliquots were pipetted out into separate10 ml volumetric flasks, and dilutions were made with distilled water to obtain working standard solutions of concentrations ranging from 1 to 100 µg/ml. The absorbance of these solutions was measured at 274 nm for UDB and at 287 nm for TFC. For the standard solutions, the analytical concentration range was found to be 20-80 µg/ml, and those values are given in Table 5.7.  

Calibration curve for the UDB and TFC(20–80 µg/ml):  

Appropriate volumes of aliquots from the standard UDB and TFC stock solutions were transferred to different volumetric flasks of 10 ml capacity. The volumes were adjusted to the mark with distilled water to obtain concentrations of 20, 30, 40, 50, 60, 70, and 80 µg/ml for both UDB and TFC. The absorbance spectra of each UDB solution against distilled water as a blankweremeasuredat274nm, and for each TFC solution, the absorbance was measured at 287 nm. The graphs of absorbance against concentration were plotted and are shown in Fig. 5.3. The regression equations and correlation coefficients were determined and reported in Table 5.8.  

Sample preparation for determination of UDB and TFC from dosage form:  Twenty tablets of two brands, each containing 100 mg of UDB and TFC, were weighed and finely powdered. The powder equivalent to 100 mg of each drug was accurately weighed and transferred to separate 100 ml volumetric flasks containing 25 ml of distilled water and sonicated for 5 minutes. The flasks were shaken and the volumes were made up to the mark with distilled water to give solutions of 1000 µg/ml for both UDB and TFC. These solutions were centrifuged at 2000 rpm for10minutesand carefully filtered through Whatman filter paper(No.41).From each solution, 10 ml was pipetted and diluted to 50 ml with distilled water to give solutions of 200 µg/ml, which were used for the estimation of UDB and TFC, respectively.  

Validation of Spectrophotometric method:  

All the parameters are same as Method A.  

ANALYTICAL METHOD DEVELOPMENT  

Various new analytical methods are required for controlling the quality of constantly growing new drugs. Alternate methods for existing (non-Pharmacopoeia) products are developed to reduce the cost and time for better precision and ruggedness. Trial runs are conducted, method is optimized and validated. When alternate method proposed is intended to replace the existing procedure comparative laboratory data including merit / demerits are made available.  

VALIDATION[18]  OBJECTIVE AND PARAMETERS OFANALYTICAL METHOD VALIDATION   

ACCURACY  

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.  

PRECISION  

The precision of an analytical procedure expresses the closeness of agreement(degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.  

REPEATABILITY  

Repeatability expresses the precision under the same operating conditions over a short interval of time.  

REPRODUCIBILITY  

The procedure is carried out by different analyst in different laboratories using different equipment, regents and laboratories setting.   

SELECTIVITY  

The selectivity of a method is a measure of how capable it is of measuring the analyte alone in the presence of other compounds contained in the sample.  

SENSITIVITY  

The sensitivity of method indicates how responsive it is to a small change in the concentration of an analyte. It   

SPECIFICITY  

An investigation of specificity should be conducted during the validation of identification tests and determination of impurities.  

LINEARITY RANGE  

The equation of a straight line takes the form.  

Y= a+ b x  

Where„a? is the intercept of  the straight with the y axis and„ b’is the slope of the line.The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.  

LIMITOFDEFECTION  

. It is formally defined as follows.  

X– Xb =3sb  

Where X?  is the signal from the sample. „Xb? is the signal from the analytical blank and Sb is the SD of the reading for the analytical blank. The detection limit is usually expressed as the concentration of analyte(percentage parts per million)in the sample. We can calculate LOD by using the following formula.  

LOD=3*SD /slope of calibration curve SD =Standard deviation of intercepts.  

LIMITOFQUANTITATION  

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.  

ROBUSTNESS  

This term refers to how resistant the precision and accuracy of an assay is to small variation in the method ,e.g.changes of instrumentation, slight variation in extraction procedure, sensitivity to minor impurities in reagents, etc.  

RUGGEDNESS  

Ruggedness is a measurement of reproducibility of test results under the variation in condition normally expected from laboratory to laboratory and from analyst to analyst.   

VALIDATION OF ANALYTICAL METHOD:   

1.Accuracy  

2.Precision  

3.Linearity  

4.Limit of detection(LOD) 5.Limit of quantitation(LOQ)  

6.Ruggedness:  

7.Robustness:  

RESULT AND DISCUSSION  

 TRAILS FOR METHOD DEVELOPMENT

Trail 1:

Mobile phase       : Methanol: Water (80:20%v/v)

            Column                : X-Bridge (4.6 ×150mm, 5µm particle size) Make; waters

Flow rate              : 2.0ml/min Wavelength       : 252nm Column temp       : 30ºC  Injection Volume : 10µl Run time         :  5 minutes

Fig. 10 (a) :- Chromatogram for Trail 1

Table 12 (a) :- Peak Results for Trail 1

Observation: This trial shows improper separation of sample peaks and less plate count, improper baseline in the chromatogram. So more trials were required for obtaining good peaks.

Trail 2:

Mobile phase  :  Methanol: Acetonitrile (40:60 v/v)

            Column                : Hypersil C18 (4.6mm×250mm) 5µ Particle Size 

Flow rate              : 0.9 ml/min Wavelength       : 246nm Column temp       : Ambient Injection Volume : 5µl Run time           : 10 minutes

Fig. 10 (b) :- Chromatogram for Trail 2

Table 12 (b) :- Peak Results for Trail 2

 Observation: From the above chromatogram it was observed that the baseline is improper      and sample peaks are not well separated. So it requires more trials to obtain well peaks.

 Trail 3:

Mobile phas       : Methanol: Water (60:40 % v/v)  

            Column                :  Symmetry C18 (4.6×250mm 5µm)

Flow rate              : 0.9 ml/min Wavelength       : 242 nm

Column temp       : 33ºC Injection Volume : 10µl  Run time              : 6 minutes

Fig. 10 (c) :- Chromatogram for Trail 3

Table 12 (c) :- Peak Results for Trail 3

Observation: This trial show very less plate count and sample peaks are not well separated,  so more trials were required for obtaining good peaks.

PART A: DERIVATIVE SPECTROSCOPY

METHOD A: FIRST ORDER DERIVATIVE SPECTROSCOPY

Table: 5.1. Results of Calibration curve at 267 nm for UDB and TFC by First order Derivative Spectroscopy

Table: 5.2. Optimum conditions, Optical characteristics and Statistical data of the Regression equation in First order Derivative Spectroscopy

Table: 5.3. Determination of Accuracy results of UDB and TFC by First order Derivative Spectroscopy

**Average of six determinations  Tablet 1: RINVOQ Tablet 2: REMATIB

Table: 5.4. Determination of Precision results for UDB at 267 nm by First order Derivative Spectroscopy

Fig: (5.2). Calibration curve for UDB and TFC at 267 nm by First order Derivative Spectroscopy

Table: 5.5. Repeatability data for UDB and TFC at 267 nm by First Order Derivative Spectroscopy

Table: 5.6. Ruggedness results for UDB and TFC at 267 nm by First order Derivative Spectroscopy

** Average of six determinations.  Tablet 1: RINVOQ Tablet 2: REMATIB

METHOD B : SECOND ORDER DERIVATIVE SPECTROSCOPY

Table: 5.7. Results of Calibration curve at 274 nm for UDB and TFC by Second order Derivative Spectroscopy

Table: 5.8. Optimum conditions, Optical characteristics and Statistical data of the Regression equation in Second order Derivative Spectroscopy

*Y= b C + a where C is the concentration of UDB in µg / ml and Y is the absorbance at the respective lmax.

**Average of six determinations.

Table: 5.9. Determination of Accuracy results for UDB and TFC by Second order Derivative Spectroscopy

Table: 5.9. Determination of Accuracy results for UDB and TFC by Second order Derivative Spectroscopy

Table: 5.10. Determination of Precision results for UDB and TFC at 274 nm by Second order Derivative Spectroscopy

Fig: (5.4). Calibration curve of Linearity for UDB and TFC at 274 nm by Second order Derivative Spectroscopy

Table: 5.11. Repeatability data for UDB and TFC at 274 nm by Second order Derivative Spectroscopy

**Average of six determinations

Table: 5.12. Ruggedness results for UDB and TFC at 274 nm by Second order Derivative Spectroscopy

** Average of six determinations  Tablet 1: RINVOQ Tablet 2: REMATIB

PART B: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

VALIDATION OF ANALYTICAL METHOD:

Validation of an analytical method is the process to establish by laboratory studies that the performance characteristic of the method meets the requirements for the intended analytical application. Performance characteristics are expressed in terms of analytical parameters.

 ACCURACY:

The accuracy of the method will be inferred by establishing the precision and linearity of standard.

Table: 5.15. Accuracy results for UDB and TFC (for dosage form)

2. PRECISION:

The precision of the analytical method was studied by analysis of multiple sampling of homogeneous sample. The precision expressed as standard deviation or relative standard deviation.

 Method precision (for pure drug)

Table: 5.17. Method Precision results for UDB and TFC (for pure drug)

** Average of five determinations

Table: 5.18. Method Precision results for UDB and TFC (for dosage form)

Table: 5.19. Linearity results for UDB and TFC

LIMIT OF QUANTITATION (LOQ):

Table: 5.22: LOQ results of UDB and TFC

4.RUGGEDNESs

The ruggedness of an analytical method is determined by analysis of aliquots from homogenous lots by different analysts using operational and environmental conditions that may differ but are still within the specified parameters of the assay. The assay of UDB was performed by different analyst and on different dates (days).

Table: 5.23: Ruggedness results of UDB and TFC (Day-1, Analyst-1)

Table: 5.24. Ruggedness results of UDB and TFC (Day-2, Analyst- 2)

  4. ROBUSTNESS :- 

The robustness of an analytical method is determined by analysis of aliquots from homogenous lots by differing physical parameters that may differ but are still within the specified parameters of the assay.

Table: 5.25. (a) Chromatographic Condition: Change in flow rate

Table: 5.26. Robustness results of UDB and TFC (Flow rate -1.6)

Table: 5.27. Robustness results of UDB (Flow rate -1.4)

Table: 5.28. (b) Chromatographic Condition: Change in mobile phase ratio

Table: 5.29. Robustness results of UDB and TFC                          ( Triflouroacetic acid: Acetonitrile = 82 : 18, v / v)

Table: 5.30. Robustness results of UDB and TFC

(Triflouroacetic acid: Acetonitrile = 80 : 20, v / v)

Table: 5.30. Robusness results of UDB and

TFC (Triflouroacetic acid:Acetonitrile=80:20,v/ v