Int J App Pharm, Vol 17, Issue 4, 2025, 420-434Original Article
A LIQUID CHROMATOGRAPHIC METHOD FOR THE RELIABLE QUANTIFICATION OF UPADACITINIB AND ITS SPECIFIED IMPURITIES
SUBHASHINI KANTHETI1, RUDRARAJU RAMESH RAJU2*, GIRI PRASAD GORUMUTCHU3
1,2*Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Andhra Pradesh, India. 3Department of Chemistry, A.G. and S.G. Siddhartha Degree College of Arts and Science, Vuyyuru-521165 Andhra Pradesh, India
*Corresponding author: Rudraraju Ramesh Raju; *Email: rrraju1@gmail.com
Received: 20 Feb 2025, Revised and Accepted: 17 May 2025
ABSTRACT
Objective: In the proposed investigation, a novel RP-UPLC technique for the simultaneous measurement of Upadacitinib and its impurities is developed. The technique's suitability for both tablet and bulk medication dose forms was confirmed.
Methods: A mobile phase consisting of 2.5 g of sodim hexane-1-sulphonic acid in 1 L of water was used to achieve an isocratic elution. The pH was adjusted to 3.0 using diluted formic acid: methanol (60:40) at a flow rate of 1 ml. min-1, using an X-Bridge Phenyl (250 x 4.6 mm, 5 µm) column.
Results: Sharp peaks of Upadacitinib and its impurities were detected at 6.730, 2.823, 3.844, 4.872, 9.991, 10.499, and 10.932 min, respectively, at 256 nm as the peak using the PDA detector. Among other aspects of system suitability, USP resolution is greater than or equal to 2, USP plate count exceeds 4000, and USP tailing is good. Purity Flag "No" means that the peak is uniform (provided by Empower program). If the purity angle is smaller than the purity threshold, the peak is homogeneous. The procedure was verified in accordance with the ICH recommendations. The procedure was validated in accordance with ICH recommendations. The concentration ranges of Upadacitinib (75–450 µg/ml), impurities 1, 2, 4, and 5 (2.50–15.00 µg/ml), and impurities 3 and 6 (1.25–7.50 µg/ml) were all subjected to linear regression for the calibration curve. LOQ (34.41, 0.057, 0.211, 0.181, 0.151, 0.377, and 0.165 µg ml–1) and LOD (11.35, 0.019, 0.070, 0.060, 0.049, 0.124, and 0.055 µg ml–1) for and Upadacitinib and impurities, respectively. Accuracy, precision, and resilience were among the other confirmed characteristics that were within acceptable ranges.
Conclusion: As a result, this method was chosen for shared analysis. Finally, the restrictions of the system-appropriate parameters and validation parameters are acceptable. Stability-indicating experiments under various stress conditions were successfully included to the approach.
Keywords: Upadacitinib, Impurities, UPLC, Empower software and PDA detector
© 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2025v17i4.54010 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
The regular name of Upadacitinib (UDB) is Rinvoq in the form of tablet dosage. UDB is used in the treatment of psoriatic arthritis, rheumatoid arthritis, ulcerative colitis, atopic dermatitis, axial spondyloarthritis, Crohn's disease, and ankylosing spondylitis [1-3]. UDB was accepted by the states, the European Union, and the United States in 2019 [4-6]. Upadacitinib is an inhibitor of Janus Kinase (JAK) that works by blocking the action of enzymes called (JAK) Janus Kinases. These enzymes play a role in initiating processes that result in inflammation; inhibiting their impact reduces inflammation in the joints [2, 6]. Upper respiratory tract infections (sinuses, common colds), nausea, coughing, and fever are common adverse effects [6].
Upadacitinib quantities in the body are raised by drugs that potently inhibit the liver enzyme CYP3A4, such as clarithromycin, ketoconazole, and itraconazole. Ketoconazole elevated the AUC by 75% in one study. On the other hand, drugs that significantly activate CYP3A4 decrease the levels of Upadacitinib [6, 7]. Upadacitinib has a solubility of about 30 mg/ml in organic solvents such as Dimethyl Sulphoxoide (DMSO) and dimethyl form amide. Upadacitinib has limited solubility in aqueous solutions. Upadacitinib can be dissolved in DMSO first, and then diluted with the preferred aqueous buffer to optimize its solubility in aqueous buffers [8].
Formulation Development for upadacitinib extended-release tablets using in vitro-in vivo correlation [9]. UPLC-MS/MS method development for the pharmacokinetics study and quantitative analysis of upadacitinib in beagle dog plasma [10]. Associated substances axitinib, zanubrutinib, and upadacitinib method development and validation using RP-HPLC, and Its degradation products were characterized using LC-MS/MS [11]. Pharmacokinetic application of concurrent UPLC-MS/MS measurement of methotrexate and upadacitinib in rat plasma [12]. The goal of the current work was to develop a new UPLC method with a PDA detector and validate the method according to ICH Guidelines, which was successfully employed to study the pharmacokinetics of Upadacitinib and its related impurities (fig. 1).
MATERIALS AND METHODS
Materials
Merck Chemicals in Mumbai supplied the analytical and HPLC quality solvents. The likely impurity standards for the drug Upadacitinib were submitted by SJS Pharmaceuticals Lab in Hyderabad.
Instruments
Digital pH meters (Elico LI-120) and balances (DENVER brand, SI234 model; Shimadzu AUX-220) were used to weigh and measure the items.
UPLC: An Agilent 1290 Infinity II LC System (Pump: Quaternary; Software: Empower 2.0) with a PDA detector was used.
General procedures
Standard drug (Upadacitinib) preparation
A volumetric (50 ml) flask was filled with 50 mg of Upadacitinib after it had been precisely weighed. Then, it was properly dissolved in acetonitrile, sonicated for about 15 min, and diluted to the appropriate level using a comparable solvent. The stock (1000 µg ml–1) solution was then filtered through a 0.22 µm membrane filter to remove any undissolved particles. This ensured that the final solution was clear and suitable for subsequent analytical procedures) was used to create the obtained solution. To use the solution again, dilute it properly.
Fig. 1: Upadacitinib and its related impurities
Stock solution-A (from impurities)
A 100 ml volumetric flask was filled with 5 mg of Imp-1, Imp-2, Imp-4, and Imp-5, which had been precisely weighed. After properly dissolving in acetonitrile, sonicate for approximately fifteen minutes.
Stock solution-B (from impurities)
5 mg of Imp-3 and Imp-6 were precisely weighed and then added to a 100 ml volumetric flask. After properly dissolving in acetonitrile, sonicate for approximately fifteen minutes.
Stock solution (Impurity) preparation
Add 1 ml of each of the impurity stocks A and B to a 50 ml volumetric flask of diluents.
Forced degradation and method validation studies
According to the most recent guidelines, the final optimum conditions are verified [13]. Stress samples were suitably diluted to provide the closing concentration while maintaining the intended technique conditions (50 µg/ml of Upadacitinib), and they were correlated with blank and standard chromatograms.
RESULTS
Analytical method development and optimisation
Selection of diluent
According to USP general requirements, diluent is used to create standards, sample solutions, impurity standards, placebo solutions, and system suitability solutions. These solutions were selected based on solubility experiments carried out for Upadacitinib (table 1).
Table 1: Solubility study (25 °C) of upadacitinib
| Solvent | Solubility |
| Water (H2O) | Insoluble |
| Acetone (CH3COCH3) | Slightly soluble |
| Ethyl acetate | Slightly soluble |
| Methanol | Completely Soluble |
| Acetonitrile | Completely Soluble |
In acetonitrile and methanol, the active ingredient dissolves easily, according to solubility tests. According to solubility studies, compatibility of the mobile phase, and excipient solubility, all of the solutions were prepared using acetonitrile as a diluents.
Determination of detection wavelength
First, methanol and acetonitrile solvents are chosen as mobile phase solvents based on the solubility of Upadacitinib and impurities. Analyte pKa value, polarity, and organic phase concentration were the main factors in the selection of the mobile phase. For simultaneous measurement of Upadacitinib and impurities, the Photodiode Array detector (PDA) wavelength was adjusted at 256 nm (fig. 2) at the iso-absorption point based on the absorption maxima seen for Upadacitinib and impurities.
Fig. 2: Spectra of UDB and their impurities (PDA)
Choice of chromatographic situations (Method development)
Using Waters brand HPLC instruments (Alliance model No. e2695; software-Empower 2.0), the current investigation aims to create methodology and validation. Normally (standard) solutions of Upadacitinib and its six contaminants are prepared by diluting the drug with acetonitrile.
The major important solvent of the mobile phase (MP) was chosen to be acetonitrile (LC grade), as it outperforms the other solvents (polar) in the following areas. 1). The lowest transmission density (absorbance) at shorter wavelengths (λmax) results in less sound when UV detection is performed. It advises conducting a more sensitive assessment at shorter UV wavelengths (λmax). 2) For gradient baselines, less ghost cresting is observed. Viscosity and condensed back pressure result in a much larger peak form. (5) The elution strength of acetonitrile (CAN) based solutions is higher than that of methanol (CH3CH2OH)-based solutions. 6) Using acetonitrile at modest mixture ratios, one can achieve the same retention duration with less than half the methanol (CH3CH2OH) ratio [14]. Upadacitinib is a chemical that has heterocyclic rings (imidazole ring) and one functional group (diamide group). The first is heterocyclic rings, which have a polar character, strong basicity, and are soluble in water [15]. The second is the diamide group, which is nonpolar, less basic, and less soluble in water. Based on the second point, the chemical has a nonpolar nature and is insoluble in water.
The first phase of trials was conducted using a Zorbax SB C18 250x4.6 mm, 5µ column, and mobile phase is 0.01% TFA in water (pH: 3.0): Acetonitrile (30:70, 50:50); this two trials baseline drift and unknown peaks are observed (fig. 3).
Fig. 3: Chromatograms obtained by TFA (pH: 3.0): Acetonitrile (30:70, 50:50)
The second phase of trials were conducted using Zorbax SB C18 250x4.6 mm, 5µ column and mobile phase 1.36 gm KH2PO4 in 1l water (pH: 3.0) adjested with OPA: acetonitrile (50:50), these trial, baseline drift and unknown peaks were observed (fig. 4).
Fig. 4: Chromatograms obtained by KH2PO4 (pH: 3.0): Acetonitrile (50:50)
The third phase of trials (table 2) was conducted using a Zorbax SB C18 250x4.6 mm, 5µ column and the mobile phase is 1.36 gm KH2PO4 in 1liter water (pH: 3.0) adjusted with OPA: Methanol (50:50), trial, base line drift and unknown peaks are observed (fig. 5).
Fig. 5: Chromatograms obtained by KH2PO4 (pH: 3.0): Methanol (50:50)
The fourth phase of trials (table 2) was conducted using a Zorbax SB C18 250x4.6 mm, 5µ column and the mobile phase is 1.36 gm KH2PO4 in 1l water (pH: 3.0) adjusted with OPA: (Acetonitirile+Methanol (50+50)) (60:40; 40:60), these two trials impurity peaks are not clear, and unknown peaks are observed (fig. 6).
The fifth phase of trials (table 2) was conducted using a Zorbax SB C18 250x4.6 mm, 5µ column and the mobile phase is 770.8 mg of ammonium acetate in 1l water adjust pH-3.0 with Acetic acid: Acetonitirile (50+50; 70:20) these two trials resolutions not within the limit, and unknown peaks are observed (fig. 7).
Fig. 6: Chromatograms obtained from KH2PO4: ((ACN+CH3OH) (50+50)) (60:40; 40:60)
Fig. 7: Chromatograms obtained from KH2PO4: Acetonitirile+Methanol (50:50; 70:20)
Fig. 8: Chromatograms obtained by ammonium acetate (pH: 3.0): Methanol (50:50)
The sixth phase of trials (table 2) was conducted using Waters X-Bridge Phenyl 250x4.6 mm, 5µ, column and the mobile phase is 770.8 mg of ammonium acetate in 1 L water adjustd pH-3.0 with Acetic acid: Methanol (50+50) these two trials resolutions not within the limit, and unknown peaks are observed (fig. 8).
The seventh phase of trials (table 2) was conducted using Waters X-Bridge Phenyl 250x4.6 mm, 5µ, column and the mobile phase is 2.5 g sodim Hexane-1-sulphonic acid in 1 L water adjust pH-3.0 with Dilted formic acid: Acetonitrile (30:70, 50:50, 40:60) these three trials resolutions not within the limit, and unknown peaks are observed (fig. 9).
The eight phase of trial (table 2) was conducted using Waters X-Bridge Phenyl 250x4.6 mm, 5µ, column and the mobile phase is 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Diluted formic acid: Methanol (40:60) these three trials resolutions not within the limit, and unknown peaks are observed (fig. 10).
Fig. 9: Chromatograms obtained from sodium hexane-1-sulphonic acid: Acetonitirile (30:70, 50:50, 40:60)
Fig. 10: Chromatograms obtained by sodium hexane-1-sulphonic acid: methanol (40:60)
Fig. 11: Optimized chromatograms obtained by sodium hexane-1-sulphonic acid (pH: 3.0): Methanol (60:40, v/v)
The final phase of the trial (table 2) was conducted using a Waters X-Bridge Phenyl 250x4.6 mm, 5µ, column and the mobile phase is 2.5 g sodium hexane-1-sulphonic acid in 1 l water adjusting the pH-3.0 with diluted formic acid: methanol (60:40) this trial was followed by a system suitable for conditions (fig. 11). USP tail factors (acceptable value equal to 2 or less than 2) of drug and its impurities values are 1.05, 1.25, 1.35, 1.58, 1.36,1.24, and 0.69. USP plate count (acceptable value more than 4000) of the drug and its impurities values are 8058, 2225,4768,5748,56921,61902, 68306. USP resolutions (acceptable more than 2) of the drug and its impurities (imp-1, imp-3, imp-4, imp-5, and imp-6) values 6.69, 4.54, 4.87, 13.24, 3.36 and 2.57. Finally, the method is accepted according to ICH rules (table 3).
Table 2: Conducted trails
| S. No. | Mobile phase | Column | Observation | Diluent |
| 1 | Water pH-3.0 with TFA: Acetonitrile (30:70) | Zorbax SB C18 250x4.6 mm, 5µ | Baseline not sufficient | Acetonitrile |
| 2 | Water pH-3.0 with TFA: Acetonitrile (50:50) | Zorbax SB C18 250x4.6 mm, 5µ | Baseline not sufficient | Acetonitrile |
| 3 | 1.36 g KH2PO4 in 1l water adjust pH-3.0 with OPA: Acetonitrile (50:50) | Zorbax SB C18 250x4.6 mm, 5µ | Unknown peaks observed | Acetonitrile |
| 4 | 1.36 g KH2PO4 in 1l water adjust pH-3.0 with OPA: Methanol (50:50) | Zorbax SB C18 250x4.6 mm, 5µ | peaks are not separated | Acetonitrile |
| 5 | 1.36 g KH2PO4 in 1l water adjust pH-3.0 with OPA: Acetonitirile+Methanol (50+50) (60:40) | Zorbax SB C18 250x4.6 mm, 5µ | Imp-2 peak is not clear | Acetonitrile |
| 6 | 1.36 g KH2PO4 in 1l water adjust pH-3.0 with OPA: acetonitirile+Methanol (50+50) (50:40) | Zorbax SB C18 250x4.6 mm, 5µ | Imp-4and5 resolution not good | Acetonitrile |
| 7 | 770.8 mg of ammonium acetate in 1l water adjust pH-3.0 with Acetic acid: Acetonitirile (50:50) | Zorbax SB C18 250x4.6 mm, 5µ | Upadacitinb and Imp-4 resolution not good | Acetonitrile |
| 8 | 770.8 mg of ammonium acetate in 1l water adjust pH-3.0 with Acetic acid: Acetonitirile (70:20) | Zorbax SB C18 250x4.6 mm, 5µ | Imp-6 not eluted | Acetonitrile |
| 9 | 770.8 mg of ammonium acetate in 1l water adjust pH-3.0 with Acetic acid: Methanol (50:50) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | Peaks are not good | Methanol |
| 10 | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Acetonitrile (30:70) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | Imp-4and5 resolution not good | Acetonitrile |
| 11 | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Acetonitrile (50:50) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | Peaks are separed | Acetonitrile |
| 12 | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Acetonitrile (40:60) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | Peaks are separed | Acetonitrile |
| 13 | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Methonol (40:60) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | Peaks are separed; concentration finalize | Methanol |
| 14 | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Methonol (60:40) | Waters X-Bridge Phenyl 250x4.6 mm, 5µ | This method is suitable for validation | Methanol |
Table 3: Method conditions
| Parameter | Condition |
| Diluent | Methanol |
| Detector wavelength | 256 nm |
| Run time | 15 min |
| Column temperature | Ambient |
| Test temperature | Ambient |
| Injection volume | 10µl* |
| Flow rate | 1 ml min–1 |
| Column | X-Bridge Phenyl (250 mm x 4.6 mm, 5µm) |
| Mode of separation | Isocratic |
| Mobile Phase | 2.5 g sodim Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with Dilted formic acid: Methanol (60:40) |
DISCUSSION
The HPLC process was designed and improved in accordance with ICH (2005) requirements (Q2R1) [13]. Each verified parameter is covered in depth in the section below.
Optimal conditions for the technique System appropriateness parameters are listed in table 4. For Upadacitinib (UDB) and its impurities, the USP tailing factor is 0.69–1.58, indicating that the peaks have precise Gaussian forms and are symmetrical around their axis. By achieving a good resolution between the neighboring peaks, the USP plate count (68306-2225) demonstrates that the selected column is successful in resolving the sample components (fig. 12). Six Upadacitinib (UDB) injections were made in the system precision study, and the percentage RSD was determined to be 0.108 (<1%). This indicates that the amounts produced are accurate. Chromatograms for system appropriateness and system precision are displayed in table 5 and table 6.
A Diluent solution was made using the revised technique and added to the chromatographic apparatus (fig. 13). The retention periods of the active and impurity peaks were not interfering because of the diluents.
To create a placebo solution using the enhanced technique and add it to the chromatographic apparatus (fig. 13). The retention durations of the active and impurity peaks were unaffected by the placebo.
Fig. 12: System suitability chromatogram
Fig. 13: Chromatogram obtained by blank and placebo
Table 4: Parameters of system suitability
| S. No. | Parameter | Parameter values | |||||
| UDB | Imp-1 | Imp-2 | Imp-3 | Imp-4 | Imp-5 | ||
| 1 | Retention time (min) | 6.730 | 2.823 | 3.844 | 4.872 | 9.991 | 10.499 |
| 2 | Peak area (µV*Sec) | 11638823 | 900850 | 938896 | 357027 | 817722 | 763990 |
| 3 | USP tailing | 1.05 | 1.25 | 1.35 | 1.58 | 1.36 | 1.24 |
| 4 | USP plate count | 8058 | 2225 | 4768 | 5748 | 56921 | 61902 |
| 5 | USP resolution | 6.69 | ----- | 4.54 | 4.87 | 13.24 | 3.36 |
| 6 | Purity1 angle | 0.028 | 0.056 | 0.037 | 0.567 | 0.137 | 1.158 |
| 7 | Purity1threshold | 5.049 | 5.027 | 5.015 | 5.049 | 5.019 | 5.020 |
| 8 | Purity1 flag | No | No | No | No | No | No |
| The Purity Flag "No" indicates that the peak is homogeneous (provided by Empower program). Should the purity angle be below the purity criterion, the peak is homogeneous? |
|||||||
Table 5: System precision
| S. No. | UDB | IMP-1 | IMP-2 | IMP-3 | IMP-4 | IMP-5 | IMP-6 |
| 1 | 11654278 | 907451 | 936521 | 356241 | 815462 | 765421 | 524685 |
| 2 | 11452174 | 902365 | 935214 | 352647 | 816532 | 765847 | 524758 |
| 3 | 11320658 | 902451 | 934588 | 356582 | 817458 | 762438 | 526321 |
| 4 | 11526489 | 905526 | 936548 | 353625 | 815263 | 764985 | 525898 |
| 5 | 11847548 | 905462 | 933365 | 354251 | 816632 | 767845 | 525461 |
| 6 | 11745826 | 901174 | 937451 | 355824 | 814965 | 763299 | 520965 |
| Mean | 11591162 | 904071.5 | 935614.5 | 354861.7 | 816052 | 764972.5 | 524681.3 |
| S. D. | 178096.9 | 2213.736 | 1374.788 | 1448.86 | 884.67 | 1753.17 | 1760.38 |
| % RSD | 1.536 | 0.245 | 0.147 | 0.408 | 0.108 | 0.229 | 0.336 |
Every peak's retention time can be used to establish whether or not all of the peaks are well-defined in relation to one another. When the resolution between two eluting crests is more than two, it indicates that the crests are well separated.
To find the peak purity, the purity angle and purity threshold are compared. The purity angle provides information about the solvent angle and purity noise angle together. The spectral homogeneity, represented by the purity flag, is stretched when the purity angle and purity threshold are compared [16]. Peak purity assesses a crest's spectral homogeneity.
In the present study, the peak purity of the chromatograms obtained from spiked tasters was estimated using Waters Empower Networking Computer software. Purity angle values and purity threshold values are used to assess both the spectrum homogeneity and peak purity [17]. If the purity threshold is exceeded by the peak purity angle, the analyzed peak is homogeneous (fig. 14-18).
Fig. 14: Typical chromatograms of unspiked sample and purity plot of UDB
Fig. 15: Chromatograms of sample spiked with impurities purity plot for UDB
Table 6: System precision parameters
| UDB | ||||||
| S. No. | RT | Plate count | Resolution | Tail factor | Peak purity | Purity angle |
| 1 | 6.734 | 8067 | 6.55 | 1.06 | 5.027 | 0.058 |
| 2 | 6.735 | 8066 | 6.57 | 1.24 | 5.048 | 0.096 |
| 3 | 6.734 | 8067 | 6.63 | 1.52 | 5.025 | 0.048 |
| 4 | 6.733 | 8078 | 6.47 | 1.39 | 5.087 | 0.039 |
| 5 | 6.732 | 8036 | 6.44 | 1.25 | 5.048 | 0.069 |
| 6 | 6.733 | 8025 | 6.14 | 1.25 | 5.019 | 0.037 |
| IMP-1 | ||||||
| 1 | 2.821 | 2209 | 1.05 | 5.028 | 0.065 | |
| 2 | 2.823 | 2238 | 1.04 | 5.015 | 0.025 | |
| 3 | 2.824 | 2236 | 1.25 | 5.057 | 0.036 | |
| 4 | 2.826 | 2269 | 1.04 | 5.027 | 0.064 | |
| 5 | 2.827 | 2258 | 1.69 | 5.021 | 0.067 | |
| 6 | 2.826 | 2265 | 1.78 | 5.014 | 0.069 | |
| IMP-2 | ||||||
| 1 | 3.843 | 4739 | 4.33 | 1.28 | 5.027 | 0.025 |
| 2 | 3.842 | 4735 | 4.35 | 1.36 | 5.048 | 0.096 |
| 3 | 3.843 | 4758 | 4.56 | 1.52 | 5.096 | 0.048 |
| 4 | 3.844 | 4758 | 4.85 | 1.06 | 5.034 | 0.089 |
| 5 | 3.843 | 4768 | 4.86 | 1.34 | 5.048 | 0.067 |
| 6 | 3.844 | 4758 | 4.56 | 1.69 | 5.018 | 0.067 |
| IMP-3 | ||||||
| 1 | 4.871 | 5792 | 4.29 | 1.37 | 5.015 | 0.539 |
| 2 | 4.872 | 5796 | 4.25 | 1.41 | 5.048 | 0.585 |
| 3 | 4.871 | 5764 | 4.20 | 1.36 | 5.068 | 0.578 |
| 4 | 4.872 | 5725 | 4.57 | 1.25 | 5.018 | 0.579 |
| 5 | 4.871 | 5724 | 4.55 | 1.14 | 5.015 | 0.558 |
| 6 | 4.872 | 5747 | 4.85 | 1.48 | 5.049 | 0.537 |
| IMP-4 | ||||||
| 1 | 9.991 | 56987 | 13.69 | 1.41 | 5.048 | 0.158 |
| 2 | 9.990 | 56968 | 13.65 | 1.30 | 5.096 | 0.157 |
| 3 | 9.992 | 56924 | 13.58 | 1.38 | 5.041 | 0.167 |
| 4 | 9.994 | 56965 | 13.54 | 1.67 | 5.018 | 0.164 |
| 5 | 9.993 | 56954 | 13.58 | 1.01 | 5.024 | 0.149 |
| 6 | 9.995 | 56698 | 13.25 | 1.08 | 5.019 | 0.125 |
| IMP-5 | ||||||
| 1 | 10.497 | 61867 | 3.05 | 1.24 | 5.069 | 1.121 |
| 2 | 10.496 | 61856 | 3.09 | 1.22 | 5.037 | 1.148 |
| 3 | 10.497 | 61598 | 3.07 | 1.20 | 5.039 | 1.107 |
| 4 | 10.495 | 61587 | 3.09 | 1.45 | 5.005 | 1.131 |
| 5 | 10.494 | 61527 | 3.01 | 1.27 | 5.063 | 1.103 |
| 6 | 10. 93 | 61478 | 3.69 | 1.37 | 5.019 | 1.137 |
| IMP-6 | ||||||
| 1 | 10.935 | 68258 | 2.59 | 0.96 | 5.027 | 0.137 |
| 2 | 10.936 | 68358 | 2.57 | 0.91 | 5.096 | 0.148 |
| 3 | 10.934 | 68364 | 2.55 | 1.91 | 5.076 | 0.149 |
| 4 | 10.935 | 68357 | 2.34 | 1.69 | 5.069 | 0.157 |
| 5 | 10.936 | 68367 | 2.37 | 1.37 | 5.020 | 0.134 |
| 6 | 10.934 | 68304 | 2.47 | 1.68 | 5.019 | 0.137 |
Initially, 6h, 12h, 18h, and 24h tests were performed for the degradation parameters (table 7) (acid, base, oxidation, hydrolysis, reduction, thermal, and photolytic (UV)). The base hydrolysis degradation, all conditions are less stable, while in thermal degradation, all conditions are more stable. The approach is exact towards Upadacitinib and impurities, as the purity threshold is higher than the purity angle in all stress parameter, which represents the studied peak, is homogenous (table 8) (fig. 19-25).
Fig. 16: Purity plot for imp-1 and imp-2 (from impurity spiked sample)
Fig. 17: Purity plot for impurity-3 and impurity-4 (from spiked impurity sample)
Fig. 18: Purity plot for impurity-5 and impurity-6 (from spiked impurity sample)
Fig. 19: Chromatograms acid stressed sample and purity plot for UDB
Fig. 20: Chromatograms base stressed sample and purity plot for UDB
Fig. 21: Chromatograms peroxide stressed sample and purity plot for UDB
Fig. 22: Chromatograms thermal stressed sample and purity plot for UDB
Fig. 23: Chromatograms UV stressed sample and purity plot for UDB
Fig. 24: Chromatograms hydrolysis stressed sample and purity plot for UDB
Fig. 25: Chromatograms reduction stressed sample and purity plot for UDB
Table 7: Degradation parameters
| Parameter | Initial degradation | 6h degradation | 12h degradation | 18h degradation | 24h degradation | ||||||||||
| Amount recovered | % assay | % deg | Amount recovered | % assay | % deg | Amount recovered | % assay | % deg | Amount recovered | % assay | % deg | Amount recovered | % assay | % deg | |
Control sample (No degradation) |
291.3202 | 97.11 | ------ | 291.3202 | 97.11 | ------- | 291.3202 | 97.11 | ------ | 291.3202 | 97.11 | ------- | 291.3202 | 97.11 | ------- |
| Acid hydrolysis | 275.6542 | 91.88 | 8.12 | 267.5515 | 89.19 | 10.82 | 252.5241 | 84.17 | 15.83 | 241.2634 | 80.42 | 19.58 | 220.7904 | 73.60 | 26.403 |
| Base hydrolysis | 275.0455 | 91.68 | 8.32 | 252.696 | 84.23 | 15.77 | 246.4281 | 82.14 | 17.86 | 235.0564 | 78.35 | 21.65 | 213.9037 | 71.30 | 28.698 |
| Oxidation | 275.2725 | 91.76 | 8.24 | 277.8712 | 92.62 | 7.38 | 275.2751 | 91.76 | 8.24 | 278.9626 | 92.99 | 7.012 | 284.1078 | 94.70 | 5.2973 |
| Hydrolysis | 275.0984 | 91. 70 | 8.30 | 275.0387 | 91.68 | 8.32 | 275.0127 | 91.67 | 8.33 | 275.7637 | 91.92 | 8.08 | 275.2725 | 91.76 | 8.2424 |
| Reduction | 275.3656 | 91.79 | 8.21 | 269.3271 | 89.78 | 10.22 | 258.3445 | 86.11 | 13.89 | 249.3793 | 83.13 | 16.87 | 235.1111 | 78.37 | 21.629 |
| Thermal | 291.7218 | 97.24 | 2.76 | 290.9422 | 96.98 | 3.02 | 290.4225 | 96.81 | 3.19 | 290.1626 | 96.72 | 3.28 | 281.3273 | 93.78 | 6.2242 |
| Photolytic (UV) | 282.1069 | 94.03 | 5.96 | 281.3273 | 93.78 | 6.22 | 280.9895 | 93.66 | 6.34 | 278.0531 | 92.68 | 7.32 | 277.3774 | 92.46 | 7.5408 |
| *B= (97.11-A)/97.11*100 | |||||||||||||||
Table 8: Purity angle and purity threshold values
| Parameter | Initial degradation | 6h degradation | 12h degradation | 18h degradation | 24h degradation | |||||
Purity angle |
Purity threshold |
Purity angle |
Purity threshold |
Purity angle |
Purity threshold |
Purity angle |
Purity threshold |
Purity angle |
Purity threshold |
|
Control sample (No degradation) |
0.028 | 5.049 | 0.028 | 5.049 | 0.028 | 5.049 | 0.028 | 5.049 | 0.028 | 5.049 |
| Acid hydrolysis | 0.068 | 5.065 | 0.054 | 5.068 | 0.065 | 5.062 | 0.021 | 5.035 | 0.524 | 5.032 |
| Base hydrolysis | 0.065 | 5.059 | 0.002 | 5.096 | 0.004 | 5.035 | 0.005 | 5.035 | 0.025 | 5.069 |
| Oxidation | 0.023 | 5.021 | 0.054 | 5.021 | 0.034 | 5.023 | 0.021 | 5.026 | 0.041 | 5.036 |
| Hydrolysis degradation | 0.024 | 5.035 | 0.046 | 5.064 | 0.065 | 5.056 | 0.048 | 5.057 | 0.048 | 5.055 |
| Reduction degradation | 0.024 | 5.059 | 0.035 | 5.035 | 0.058 | 5.035 | 0.057 | 5.095 | 0.095 | 5.048 |
| Thermal degradation | 0.064 | 5.025 | 0.064 | 5.026 | 0.035 | 5.026 | 0.031 | 5.016 | 0.031 | 5.016 |
| Photolytic degradation (UV) | 0.059 | 5.056 | 0.044 | 5.097 | 0.035 | 5.066 | 0.032 | 5.035 | 0.023 | 5.021 |
Concentration (table 9) (75–450 µg ml–1; Upadacitinib, 2.50–15.00 µg ml–1; IMP1, IMP2, IMP5, 1.25–7.50 µg ml–1; IMP3, IMP4) and detector response calibration curves were created. Fig. 26 shows the results of the linear regression technique used to evaluate the linearity. According to table 9, the selected impurities displayed values more than 0.9973, whereas the calibration curves of upadacitinib displayed correlation coefficient (R2) values greater than 0.9981. The findings show a very good linear relationship between the concentrations and peak areas produced by the proposed method.
Table 9: Linearity concentration and response peak areas
| S. No. | Con | (UDB) | Con | IMP-1 | IMP-2 | IMP-4 | IMP-5 | Con | IMP-3 | IMP-6 |
| 1 | 75 | 3393309 | 2.50 | 251457 | 250585 | 198564 | 192956 | 1.25 | 99478 | 133260 |
| 2 | 150 | 6013221 | 5.00 | 468532 | 442156 | 412036 | 365471 | 2.50 | 185469 | 265124 |
| 3 | 225 | 9425746 | 7.50 | 659837 | 678546 | 586325 | 564216 | 3.75 | 282145 | 402351 |
| 4 | 300 | 11638823 | 10.00 | 900850 | 938896 | 817722 | 763990 | 5.00 | 357027 | 520317 |
| 5 | 375 | 14865287 | 12.50 | 1114574 | 1185463 | 993265 | 946325 | 6.25 | 463259 | 648795 |
| 6 | 450 | 17842654 | 15.00 | 1315646 | 1366594 | 1205415 | 1122354 | 7.50 | 555698 | 786532 |
Table 10: Regression parameters summary
| S. No. | Parameter | Obtained values | ||||||
| UDB | Imp-1 | Imp-2 | Imp-3 | Imp-4 | Imp-5 | Imp-6 | ||
| 1 | Residual sum of squares | 0.9981 | 0.9994 | 0.9973 | 0.9986 | 0.999 | 0.9995 | 0.9996 |
| 2 | Slope | 38482 | 85715 | 92232 | 72899 | 80107 | 75307 | 103665 |
| 3 | Y-Intercept | 428240 | 35141 | 3341.7 | 4910.8 | 1287.3 | 286.07 | 5862.6 |
The accuracy of the research approach indicates how close they obtained results are to the correct values. At the description level, a rise in the measured impurity of upadacitinib was used to evaluate the recovery (accuracy) on a known quantity of placebo. In accordance with the suggested methodology, models were set up in three triplicates for the drug and its contaminants at various concentrations, namely 50%, 100%, and 150% of the target analyte (table 13). For the aforementioned drug and the designated contaminants, separate percentage recovery, standard deviation, percent relative standard deviation, and (n=3) average percent recovery were computed. Upadacitinib recovery ranged from 92.80 to 98.34 (table 11). The percentage RSD ranged from 0.378 to 1.34 (<2 %). The impurity recovery percentage ranged from 103.45 to 93.10, whereas the percentage RSD ranged from 0.116 to 1.43 (table 12). For upadacitinib and its impurities, the percentage RSD values fall well within the acceptable range. These outcomes demonstrate the method's ability to precisely separate the active ingredient and contaminants from the placebo.
Fig. 26: Calibration curves of upadacitinib and its impurities
Table 11: Results of accuracy for upadacitinib
| Level of recovery (%) | Amount added (µg ml–1) | Amount recovered (µg ml–1) | % Recovered | Statistical evaluation |
| 50 | 150 | 139.1927 | 92.79511 | %mean±SD |
| 150 | 139.8837 | 93.25583 | ||
| 150 | 140.2313 | 93.48754 | %RSD | |
| 100 | 300 | 286.3189 | 95.43962 | %mean± |
| 300 | 291.1571 | 97.05237 | SD | |
| 300 | 294.0054 | 98.00178 | %RSD | |
| 150 | 450 | 432.0561 | 96.01247 | %mean±SD |
| 450 | 442.5631 | 98.34735 | ||
| 450 | 439.528 | 97.67289 | %RSD |
Value are given in mean±SD; n=3
Table 12: Results of accuracy for impurities
| Level of recovery (%) | % Recovered | Statistical evaluation | |||||||||||
| IMP-1 | IMP-2 | IMP-3 | IMP-4 | IMP-5 | IMP-6 | IMP-1 | IMP-2 | IMP-3 | IMP-4 | IMP-5 | IMP-6 | ||
| 50 | 98.79 | 99.57 | 93.10 | 102.85 | 102.85 | 98.24 | %Mean±SD | 98.08 ±0.691 |
99.87±0.553 | 94.35±1.35 | 100.65 ±0.778 |
102.24 ±0.546 |
97.89 ±0.347 |
| 50 | 97.41 | 100.51 | 94.17 | 101.81 | 101.81 | 97.54 | |||||||
| 50 | 98.03 | 99.53 | 95.79 | 102.04 | 102.04 | 97.89 | %RSD | 0.705 | 0.554 | 1.43 | 0.773 | 0.534 | 0.355 |
| 100 | 101.18 | 101.18 | 96.39 | 101.81 | 101.81 | 100.63 | %Mean±SD | 101.17 ±0.117 |
101.07±0.282 | 96.23±0.595 | 101.60± | 101.53± | 100.19 ±0.614 |
| 100 | 101.05 | 101.28 | 95.57 | 101.58 | 101.58 | 100.45 | 0.312 | 0.305 | |||||
| 100 | 101.28 | 100.75 | 96.73 | 101.20 | 101.20 | 99.49 | %RSD | 0.116 | 0.279 | 0.619 | 0.308 | 0.300 | 0.612 |
| 150 | 101.15 | 101.11 | 96.51 | 102.16 | 102.16 | 98.87 | %Mean±SD | 102.17 ±1.17 |
101.56±0.449 | 96.91±0.357 | 101.08 ±0.879 |
101.35 ±0.878 |
99.12 ±0.248 |
| 150 | 101.89 | 101.57 | 97.18 | 100.42 | 100.42 | 99.37 | |||||||
| 150 | 103.45 | 102.01 | 97.04 | 101.48 | 101.48 | 99.13 | %RSD | 1.15 | 0.442 | 0.369 | 0.870 | 0.867 | 0.251 |
Value are given in mean±SD; n=6
Table 13: Amount recovered and added concentrations
Level of recovery (%) |
IMP-1 | IMP-2 | IMP-3 | IMP-4 | IMP-5 | IMP-6 | ||||||
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
Amount added (µg ml–1) |
Amount recovered (µg ml–1) |
|
| 50 | 5 | 4.92 | 5 | 4.98 | 2.34 | 93.10 | 5 | 4.10 | 5 | 5.14 | 2.5 | 2.46 |
| 50 | 5 | 4.87 | 5 | 5.03 | 2.35 | 94.17 | 5 | 5.03 | 5 | 5.09 | 2.5 | 2.43 |
| 50 | 5 | 4.90 | 5 | 4.98 | 2.39 | 95.79 | 5 | 5.07 | 5 | 5.10 | 2.5 | 2.45 |
| 100 | 10 | 10.13 | 10 | 10.12 | 4.82 | 96.39 | 10 | 10.19 | 10 | 10.18 | 5 | 5.03 |
| 100 | 10 | 10.10 | 10 | 10.13 | 4.79 | 95.57 | 10 | 10.17 | 10 | 10.16 | 5 | 5.02 |
| 100 | 10 | 10.13 | 10 | 10.07 | 4.84 | 96.73 | 10 | 10.13 | 10 | 10.12 | 5 | 4.97 |
| 150 | 15 | 15.17 | 15 | 15.17 | 7.24 | 96.51 | 15 | 15.16 | 15 | 15.32 | 7.5 | 7.42 |
| 150 | 15 | 15.28 | 15 | 15.24 | 7.29 | 97.18 | 15 | 15.30 | 15 | 15.06 | 7.5 | 7.45 |
| 150 | 15 | 15.56 | 15 | 15.30 | 7.28 | 97.04 | 15 | 15.03 | 15 | 15.22 | 7.5 | 7.43 |
The limit of detection (LOD) is the lowest analyte concentration that can be detected with an S/N ratio of 3:1. The limit of quantitation (LOQ) is the lowest analyte concentration that can be accurately and precisely identified using an S/N ratio of 10:1. An experimental method was used to determine the LOD and LOQ. LOQ (34.41, 0.057, 0.211, 0.181, 0.151, 0.377, and 0.165 µg ml–1) and LOD (11.35, 0.019, 0.070, 0.060, 0.049, 0.124, and 0.055 µg ml–1) for upadacitinib and impurities (IMP 1, IMP 2, IMP 3, IMP 4, IMP 5, IMP 6) were determined using accepted standards [18-26].
The stability of ordinary and sample solutions is examined from the beginning to the end of the 24-hour storage period at room temperature by using the stability methods of Shyamal et al. and Kalpana et al. [27-29]. They were administered at varying intervals, and the assay's starting and 24-hour percentages differed by less than 2%. The UDB medication is unaffected by storage conditions (table 14).
As per the intended procedure, multiple (six) samples of a comparable batch were evaluated in order to determine the method precision (MP) and intermediate precision (IP). For Upadacitinib, the average assay results in MP and IP are 91.76 and 97.63, respectively, while the impurity range is 94.74 to 102.2. The percentage RSD values of upadacitinib and its impurities vary from 2.263 to 0.057 (table 15), which is much less than the precision acceptability requirements. Furthermore, it was discovered that the system suitability parameters in precision experiments were adequate (table 16). This demonstrates the method’s accuracy.
Table 14: Results of stability (sample solutions) (RT and 2-8 °C)
| Time intervals | UDB (% assay) | % Difference | ||
| RT | 2-8 °C | RT | 2-8 °C | |
| Initial | 92.66 | 94.39 | ----- | ------ |
| 6h | 85.06 | 95.54 | 7.60 | 1.15 |
| 12h | 90.34 | 88.96 | 2.32 | 5.43 |
| 18h | 88.85 | 88.84 | 3.82 | 5.55 |
| 24h | 88.29 | 86.02 | 4.37 | 8.39 |
Table 15: Comparison of method precision (MP) and intermediate precision (IP)
| S. No. | % Assay | |||||||||||||
| Upadacitinib | Impurity 1 | Impurity 2 | Impurity 3 | Impurity 4 | Impurity 5 | Impurity 6 | ||||||||
| M. P. | I. P. | M. P. | I. P. | M. P. | I. P. | M. P. | I. P. | M. P. | I. P. | M. P. | I. P. | M. P. | I. P. | |
| 1 | 92.66 | 97.63 | 101.2 | 101.2 | 100.9 | 100.75 | 95.02 | 95.51 | 101.41 | 101.4 | 101.1 | 100.9 | 99.39 | 99.6 |
| 2 | 94.74 | 91.89 | 101.2 | 101.3 | 101.2 | 101.18 | 95.10 | 94.74 | 101.14 | 101.1 | 102.2 | 102.1 | 99.65 | 99.9 |
| 3 | 93.42 | 91.89 | 101.2 | 101.2 | 101.4 | 101.04 | 95.93 | 95.77 | 100.98 | 100.9 | 101.4 | 101.8 | 99.42 | 99.7 |
| 4 | 94.08 | 94.70 | 101.1 | 101.3 | 100.9 | 101.10 | 95.07 | 96.59 | 100.99 | 100.9 | 101.1 | 101.2 | 99.43 | 99.7 |
| 5 | 93.81 | 91.76 | 101.3 | 101.2 | 101.0 | 100.50 | 95.69 | 96.59 | 101.00 | 101.1 | 101.7 | 100.9 | 99.68 | 99.9 |
| 6 | 96.32 | 94.18 | 101.1 | 101.7 | 101.0 | 100.77 | 95.68 | 96.59 | 101.00 | 100.9 | 101.5 | 101.5 | 100.35 | 100.5 |
| Mean (n=6) | 94.174±1.147 | 93.673± | 101.2± | 101.3± | 101.1± | 100.88± | 95.42± | 95.97± | 101.09± | 101.1± 0.150 |
101.5± 0.377 |
101.4± 0.460 |
99.652± | 99.880± |
| Std. Dev | 2.119 | 0.056 | 0.184 | 0.211 | 0.223 | 0.362 | 0.698 | 0.151 | 0.330 | 0.317 | ||||
| %RSD(n=6) | 1.2177 | 2.263 | 0.057 | 0.182 | 0.209 | 0.221 | 0.380 | 0.727 | 0.149 | 0.149 | 0.372 | 0.454 | 0.332 | 0.318 |
Value are given in mean±SD; n=6
Table 16: Comparison of method precision parameters (MP) and intermediate precision parameters (IP)
System suitability sarameter |
Method precision | Intermediate precision | ||||||||||||
| UDB | IMP 1 | IMP 2 | IMP 3 | IMP 4 | IMP 5 | IMP 6 | UDB | IMP 1 | IMP 2 | IMP 3 | IMP 4 | IMP 5 | IMP 6 | |
| USP resolution | 6.02 | ------- | 4.62 | 4.52 | 13.02 | 3.36 | 2.14 | 6.48 | ------- | 4.25 | 4.69 | 13.69 | 3.48 | 2.28 |
| USP tailing factor | 1.68 | 1.08 | 1.69 | 1.48 | 1.28 | 1.48 | 0.38 | 1.24 | 1.02 | 1.36 | 1.52 | 1.58 | 1.39 | 0.21 |
| USP plate count | 8078 | 2269 | 4758 | 5747 | 56365 | 61035 | 61035 | 8025 | 2225 | 4736 | 5748 | 56364 | 61458 | 68578 |
| Purity1 angle | 0.058 | 0.065 | 0.025 | 0.539 | 0.158 | 1.121 | 0.137 | 0.058 | 0.065 | 0.025 | 0.539 | 0.158 | 1.121 | 0.137 |
| Purity1 threshold | 5.027 | 5.028 | 5.027 | 5.015 | 5.048 | 5.069 | 5.027 | 5.027 | 5.028 | 5.027 | 5.015 | 5.048 | 5.069 | 5.027 |
| Retention time (min) | 6.733 | 2.820 | 3.842 | 4.870 | 9.992 | 10.496 | 10.934 | 6.733 | 2.822 | 3.842 | 4.870 | 9.990 | 10.496 | 10.934 |
| Peak area | 11300290± 132399.2 |
902644.8± | 935395± | 352699.3± 1319.9 |
811134.7± | 764564.5± 2839.32 |
522384.3± | 11242495± 244710.6 |
903586± 1576.97 |
933737.2± 2057.65 |
354701.7± 2543.866 |
811134.7± | 764099.2± 3466.15 |
523569± |
| SD of area | 493.5408 | 1947.27 | 1208.84 | 1712.69 | 1208.84 | 1647.05 | ||||||||
| % RSD of area | 1.172 | 0.054677 | 0.208 | 0.37422 | 0.149 | 0.371 | 0.328 | 2.177 | 0.175 | 0.220 | 0.717 | 0.149 | 0.454 | 0.31458 |
| *from six standard injections | ||||||||||||||
Value are given in mean±SD, n=6
Table 17: Results of robustness/Ruggedness experiment
| Altered parameter | Altered cond. | RT (Min) | Tailing factor | Theor plates |
USP resolution | Purity threshold | Purity angle | Peak area (mean±SD) | % RSD |
| UDB | |||||||||
| Control | 6.730 | 1.05 | 8058 | 6.69 | 5.049 | 0.028 | |||
Flow (ml/min–1) |
0.7 | 8.364 | 0.28 | 14574 | 7.49 | 1.069 | 0.124 | 11250838.7±182767.89 | 1.63 |
| 1.3 | 5.726 | 1.99 | 7545 | 6.35 | 1.025 | 0.057 | 9545114±21570.14 | 0.226 | |
| Organic solvent (%) | -5 | 8.325 | 0.57 | 27629 | 9.48 | 1.018 | 0.239 | 9747763.7±19131.3 | 0.196 |
| +5 | 5.563 | 1.57 | 69424 | 5.48 | 1.035 | 0.039 | 11682572±13834.47 | 0.118 | |
| Wavelength | 250 | 8.367 | 0.95 | 14681 | 7.84 | 1.006 | 0.180 | 11371627.7±34693.2 | 0.305 |
| 262 | 8.366 | 0.57 | 14678 | 7.96 | 1.025 | 0.169 | 9553714±31114.57 | 0.326 | |
| Impurity-1 | |||||||||
| Control | 2.823 | 1.25 | 2225 | ---- | 5.027 | 0.056 | |||
Flow (ml/min–1) |
0.7 | 3.508 | 1.59 | 2558 | ---- | 1.024 | 0.069 | 1132391.7± 6080.2 | 0.537 |
| 1.3 | 2.401 | 1.02 | 2063 | ---- | 1.015 | 0.052 | 746928±1440.718 | 0.193 | |
| Organic solvent (%) | -5 | 3.151 | 1.06 | 2305 | ---- | 1.036 | 0.136 | 914148.7±1567.8 | 0.172 |
| +5 | 2.623 | 1.35 | 2135 | ----- | 1.025 | 0.058 | 894338.7±1332.41 | 0.149 | |
| Wave length | 250 | 3.509 | 1.07 | 2523 | ----- | 1.027 | 0.082 | 1124777.7±19244.8 | 1.711 |
| 262 | 3.508 | 1.67 | 2529 | ----- | 1.058 | 0.063 | 742914.7±708.9495 | 0.095 | |
| Impurity-2 | |||||||||
| Control | 3.844 | 1.35 | 4768 | 4.54 | 5.015 | 0.037 | |||
Flow (ml/min–1) |
0.7 | 4.812 | 1.68 | 5757 | 4.38 | 1.015 | 0.069 | 1159418.3±16955.8 | 1.462 |
| 1.3 | 3.242 | 1.30 | 4152 | 4.01 | 1.059 | 0.035 | 775428.7±1790.04 | 0.231 | |
| Organic solvent (%) | -5 | 4.281 | 1.57 | 5465 | 4.68 | 1.030 | 0.003 | 912381.7±3354.5 | 0.368 |
| +5 | 3.525 | 1.69 | 4359 | 4.35 | 1.015 | 0.036 | 935092±3069.21 | 0.328 | |
| Wave length | 250 | 4.811 | 1.06 | 5780 | 4.85 | 1.020 | 0.064 | 1146316±27338.12 | 2.385 |
| 262 | 4.810 | 1.59 | 5737 | 4.57 | 1.050 | 0.096 | 772525.7±3862.302 | 0.500 | |
| Impurity-3 | |||||||||
| Control | 4.872 | 1.58 | 5748 | 4.87 | 5.049 | 0.567 | |||
Flow (ml/min–1) |
0.7 | 6.094 | 1.49 | 6726 | 4.35 | 1.065 | 0.496 | 443693.3±3374.9 | 0.761 |
| 1.3 | 4.132 | 1.69 | 5254 | 4.25 | 1.069 | 0.396 | 305863.7±1915.42 | 0.626 | |
| Organic solvent (%) | -5 | 5.866 | 1.49 | 6957 | 6.25 | 1.005 | 0.869 | 342449±394.9 | 0.115 |
| +5 | 4.220 | 1.48 | 5348 | 3.86 | 1.068 | 0.335 | 332267.7±1761.6 | 0.530 | |
| Wave length | 250 | 6.095 | 1.05 | 6780 | 4.61 | 1.032 | 0.424 | 441035.7±1411.1 | 0.320 |
| 262 | 6.094 | 1.37 | 6719 | 4.67 | 1.065 | 0.436 | 304287.3±2052.14 | 0.674 | |
| Impurity-4 | |||||||||
| Control | 9.991 | 1.36 | 56921 | 13.24 | 5.019 | 0.137 | |||
Flow (ml/min–1) |
0.7 | 10.331 | 0.67 | 60284 | 8.57 | 1.085 | 0.165 | 976808.7±81.13 | 0.008 |
| 1.3 | 9.72 0 | 1.58 | 54635 | 18.65 | 1.057 | 0.169 | 715619.7±2227.72 | 0.311 | |
| Organic solvent (%) | -5 | 10.203 | 1.29 | 59244 | 10.36 | 1.007 | 0.139 | 832545.7±1984.6 | 0.238 |
| +5 | 9.77 5 | 0.69 | 54948 | 19.02 | 1.048 | 0.169 | 843421.3±3382.96 | 0.401 | |
| Wave length | 250 | 10.332 | 0.99 | 60994 | 8.81 | 1.028 | 0.181 | 976474.7±2708.17 | 0.277 |
| 262 | 10.331 | 0.69 | 60349 | 8.57 | 1.048 | 0.169 | 714898±2830.773 | 0.396 | |
| Impurity-5 | |||||||||
| Control | 10.499 | 1.24 | 61902 | 3.36 | 5.020 | 1.158 | |||
Flow (ml/min–1) |
0.7 | 10.796 | 1.25 | 65684 | 2.58 | 1.096 | 1.654 | 904170±2447.90 | 0.271 |
| 1.3 | 10.252 | 1.01 | 58574 | 3.98 | 1.024 | 0.967 | 665375.3±3099.92 | 0.466 | |
| Organic solvent (%) | -5 | 10.701 | 1.18 | 64369 | 2.15 | 1.007 | 2.439 | 795548±2393.47 | 0.301 |
| +5 | 10.33 3 | 1.18 | 59069 | 3.35 | 1.048 | 1.269 | 784105.7±2669.24 | 0.340 | |
| Wave length | 250 | 10.799 | 1.00 | 65432 | 2.78 | 1.025 | 1.602 | 906040±3189.2906 | 0.352 |
| 262 | 10.798 | 1.57 | 65579 | 2.02 | 1.048 | 1.669 | 664756.3±2847.05 | 0.428 | |
| Impurity-6 | |||||||||
| Control | 10.932 | 0.69 | 68306 | 2.57 | 5.037 | 0.115 | |||
Flow (ml/min–1) |
0.7 | 11.28 3 | 1.68 | 72320 | 2.68 | 1.036 | 0.124 | 590401±107.5034883 | 0.018 |
| 1.3 | 10.664 | 1.36 | 65698 | 2.68 | 1.024 | 0.169 | 444205.7±3384.241 | 0.762 | |
| Organic solvent (%) | -5 | 11.364 | 0.57 | 71302 | 3.57 | 1.039 | 0.129 | 514344.3±3488.25 | 0.678 |
| +5 | 10.695 | 1.27 | 65040 | 2.05 | 1.015 | 1.296 | 522490.7±1850.82 | 0.354 | |
| Wave length | 250 | 11.286 | 1.02 | 72455 | 2.89 | 1.032 | 0.120 | 593769±3207.4945 | 0.540 |
| 262 | 11.285 | 1.69 | 72579 | 2.39 | 1.048 | 0.169 | 443783±3101.223 | 0.699 | |
| Actual Condition: 2.5 g sodium Hexane-1-sulphonic acid in 1l water adjust pH-3.0 with diluted formic acid: Methanol (60:40); Wave Length: 256; Flow rate: 1 ml/min–1 |
Value are given in mean±SD; n=3
Research on robustness was done to alter the factors such as wavelength, organic content, and mobile phase flow rate. A ±3% change in flow rate, a ±5% change in organic content, and a ±10% change in flow rate were all examined various chromatographic analyses, including (n=3) %RSD, plate counts, peak regions, SD, retention duration, USP resolution, and USP tail factor (table 17). Upadacitinib's resolution with its impurities is significantly impacted by this information. The other parameters have no effect on how suitable the settings are for the system.
CONCLUSION
Following the establishment of a novel RP-UPLC isocratic technique, Upadacitinib and its designated impurities (impurities 1, 2, 3, 4, 5, and 6) were used for authentication. Among other system suitability parameters, the USP resolution is greater than or equal to 2, the USP plate count exceeds 4000, and the USP tailing is good. The resulting peaks are uniform since there is no purity flag and the purity angle is below the purity threshold. This indicates that the method is robust and reliable for the analysis of Upadacitinib, ensuring accurate quantification of both the active pharmaceutical ingredient and its impurities. Furthermore, these findings support the method's potential for quality control in pharmaceutical applications, contributing to enhanced drug safety and efficacy s technique was validated according to ICH standards, which means that all validating parameters, including robustness, accuracy, and precision, were within acceptable boundaries. As a result, this method was chosen for further studies aimed at assessing the stability of Upadacitinib under various environmental conditions. The comprehensive validation process not only reinforces the method's credibility but also paves the way for its implementation in routine testing to ensure consistent product quality. General analysis. Finally, there are reasonable restrictions on the system-appropriate and validation parameters.
ACKNOWLEDGEMENT
We thank to Principal of A. G and S. G Siddhartha Degree College of Arts and Science Vuyyuru.
FUNDING
Nil
ABBREVIATIONS
Upadacitinib (UDB), Ultra Pressure Liquid Chromatography-UPLC, Limits of Quantification (LOQ) and Limits of Detection (LOD), Janus kinase (JAK) and Dimethyl Sulphoxide (DMSO) and Photo Diode Array (PDA)
AUTHORS CONTRIBUTIONS
Subhashini Kanthti conceived the study, designed the methodology, performed the experiments and collected the data. Giri Prasad Gorumutchu analyzed the results. Rudraraju Ramesh Raju supervised the project. Subhashini Kanthti wrote the original draft, and all authors contributed to reviewing and editing the final manuscript.
CONFLICTS OF INTERESTS
The authors report no conflicts of interest.
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