Claims:I/We Claim:
1. A method of validating a specific RPLC-MS method for the determination of omadacycline in human plasma samples, wherein the method comprises:
injecting a six replicate injections of aqueous mid QC along with internal standard;
analyzing a ten lots of blank human plasma samples out of six lots free from interference were selected for assessing the selectivity, specificity;
preparing a Six LLOQ standards in screened plasma lot along with (500.00 pg/mL) and signal to noise ratio (S/N) was calculated using analyst software;
assessing an injector carryover by injecting the extracted blank samples followed by extracted ULOQ, LLOQ samples and %carry over was calculated;
preparing a calibration standards to obtain linearity range of 5.00 to 12000pg/mL and assayed in five replicates on five different days;
preparing a concentrations in a screened human plasma, wherein concentration comprises an one set of calibration standards (CS1 to CS11) and one set contains four different concentrations of quality control standards of Lower limit QC (5.00 pg/mL), Low QC (15.00 pg/mL), Mid QC (4500.00 pg/mL) and High QC (9000.00 pg/mL);
determining the extraction recovery in sextuplicate by comparing the extracted QC standards with un-extracted QC standards at three different concentrations of low (15.00 pg/mL), medium (4500.00 pg/mL), high (9000.00 pg/mL);
comparing a six extracted blank plasma samples in three replicates spiked with the un-extracted concentration of mid QC (3000.00 pg/mL) with un-extracted standards of the same concentration;
performing a dilution integrity in sextuplicate by 1:1 (DQC1) dilution and 1:4 dilution (DQC2) of 1.5 × ULOQ standard with screened blank rat plasma, wherein DQC1 and DQC2 standards were treated as un-known samples and calculated concentration was found from the standard curve regression equation; and
evaluating the re-injection stability of OM, QC samples (LQC and HQC) of P&A batch-2 samples (Pre-analyzed samples) were set aside on the bench at room temperature up to 26 hours after the initial analysis. Stability samples (RI stability samples) were re-injected after 26 hours to determine P&A of the method.

2. The method as claimed in claim 1, wherein developing and validating omadacycline using chromatography with mass spectrometry-mass spectrometry, wherein preparation of omadacycline comprises;
mixing a methanol in a range between 450 mL to 550 mL with a water in a range between 450 mL to 550 mL to form a 50% Methanol;
mixing a formic acid in a range between 50 mL to 150 mL with a water in a range between 50 mL to 150 mL to form a 0.1% formic acid;
dissolving a anhydrous sodium carbonate in a range between 25 gram to 28 gram with a water in a range between 0.5 L to 1.5 L to form a 1 M sodium carbonate;
mixing the methanol in a range between 150 mL to 250 mL with the Formic Acid in a range between 750 mL to 850 mL to from a mobile phase, wherein the mobile phase is filtered through 0.45 mL filter;
mixing a methanol in a range between 750 mL to 850 mL with a water in a range between 150 mL to 250 mL to form a Autosampler wash; and
mixing a methyl tertiary butyl in a range between 700 mL to 800 mL with a Dichloro methane in a range between 200 mL to 300 mL to form a extraction solvent

, Description:ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF OMADACYCLINE
TECHNICAL FIELD
[0001] The present disclosure relates to validating a specific RPLC-MS method for the determination of omadacycline in human plasma samples.
BACKGROUND
[0002] Methods for treating and preventing bacterial infection in a subject, methods for preparing a medicament for use in treating and preventing bacterial infection in a subject, and pharmaceutical and veterinary antimicrobial compositions.
[0003] Recently, research efforts have focused on developing new tetracycline antibiotic compositions effective under varying therapeutic conditions and routes of administration. New tetracycline analogues have also been investigated which may prove to be equal to or more effective than the originally introduced minocycline compounds.
[0004] Accordingly, a method to develop and validate a simple, selective, sensitive, rapid, rugged and reproducible assay method for the quantitative determination of Omadacycline from plasma samples
[0005] Therefor the present disclosure overcomes the above mentioned problem associated with the traditionally available system, any of the above mentioned invention can be used with the presented disclosed technique with or without modification.
OBJECTS OF THE INVENTION
[0006] It is an object of the present disclosure which validates a method with an adequate rapid, exact, specific, responsive, rugged and steady.
[0007] It is an object of the present disclosure which develop and validate a simple, selective, sensitive, rapid, rugged and reproducible assay method for the quantitative determination of Omadacycline from plasma samples.
SUMMARY
[0008] The present invention disclosures a method of validating specific RPLC-MS method for the determination of omadacycline in human plasma samples. Te method includes (i) injecting a six replicate injections of aqueous mid QC along with internal standard, 9ii) analyzing a ten lots of blank human plasma samples out of six lots free from interference were selected for assessing the selectivity, specificity, (iii) preparing a Six LLOQ standards in screened plasma lot along with (500.00 pg/mL) and signal to noise ratio (S/N) was calculated using analyst software, (iv) assessing an injector carryover by injecting the extracted blank samples followed by extracted ULOQ, LLOQ samples and %carry over was calculated, (v) preparing a calibration standards to obtain linearity range of 5.00 to 12000pg/mL and assayed in five replicates on five different days, (vi) preparing a concentrations in a screened human plasma. The concentration comprises an one set of calibration standards (CS1 to CS11) and one set contains four different concentrations of quality control standards of Lower limit QC (5.00 pg/mL), Low QC (15.00 pg/mL), Mid QC (4500.00 pg/mL) and High QC (9000.00 pg/mL), (vii) determining the extraction recovery in sextuplicate by comparing the extracted QC standards with un-extracted QC standards at three different concentrations of low (15.00 pg/mL), medium (4500.00 pg/mL), high (9000.00 pg/mL) (viii) comparing a six extracted blank plasma samples in three replicates spiked with the un-extracted concentration of mid QC (3000.00 pg/mL) with un-extracted standards of the same concentration, (ix) performing a dilution integrity in sextuplicate by 1:1 (DQC1) dilution and 1:4 dilution (DQC2) of 1.5 × ULOQ standard with screened blank rat plasma, wherein DQC1 and DQC2 standards were treated as un-known samples and calculated concentration was found from the standard curve regression equation, (x) evaluating the re-injection stability of OM, QC samples (LQC and HQC) of P&A batch-2 samples (Pre-analyzed samples) were set aside on the bench at room temperature up to 26 hours after the initial analysis. Stability samples (RI stability samples) were re-injected after 26 hours to determine P&A of the met.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 illustrates a exemplary view of developing and validating a simple, selective, sensitive, rapid, rugged and reproducible assay method for the quantitative determination of Omadacycline from plasma samples in accordance with an embodiment of the present disclosure.
[0010] FIG. 2 illustrates a exemplary view of validating specific RPLC-MS method for the determination of omadacycline in human plasma samples accordance with embodiments of the present disclosure.
[0011] FIG.3 illustrates a flow diagram of smart water management system for determining a water level in one or more storage units using a control unit and enables a warning signal to a consumer device in accordance with embodiments of the present disclosure.
DETAILED DESCRIPTION
[0012] FIG. 1 illustrates a exemplary view of developing and validating a simple, selective, sensitive, rapid, rugged and reproducible assay method for the quantitative determination of Omadacycline from plasma samples in accordance with an embodiment of the present disclosure.
[0013] The goal of this research is to develop and validate a simple, selective, sensitive, rapid, rugged and reproducible assay method for the quantitative determination of Omadacycline from plasma samples. In the way to develop a simple and easy applicable method for Omadacycline assay in human plasma for pharmacokinetic study, RPLC with MS detection was selected as the method of choice. Mass parameter Optimization, Chromatographic optimization and Extraction optimization to be optimized carefully to achieve the best results.
[0014] The mass parameter optimization was performed by direct infusion of solutions of both Omadacycline and Omadacycline-D9 into the ESI source of the mass spectrometer. Other parameters, such as the nebulizer and the heater gases and Declustering potential(DP), Entrance potential(EP),Collision energy(CE) was optimized to obtain a better spray shape, resulting in better ionization and droplet drying to form the protonated ionic Omadacycline and Omadacycline-D9 molecules. A CAD product ion spectrum for Omadacycline and Omadacycline-D9 yielded high-abundance fragment ions of mlz(amu) 456.3 and mlz(amu) 456.3 respectively (Figure 23 – Figure 24) from its parent ion mass spectra.
[0015] Initially, a mobile phase consisting of ammonium acetate and acetonitrile in varying combinations was tried, but a low response was observed. The mobile phase containing acetic acid: acetonitrile (20:80 vlv) and acetic acid: methanol (20:80 vlv) gives the better response, but poor peak shape was observed. A mobile phase of 0.1% formic acid in water in combination with methanol and acetonitrile with varying combinations were tried. Vsing a mobile phase containing 0.1% formic acid in water in combination with methanol (20:80 vlv), the best signal along with a marked improvement in the peak shape was observed for Omadacycline and Omadacycline -D9. Short length columns, such as Symmetry Shield RP18 (50mm x 2.1 mm, 3.5 m), Inertsil ODS-2V (50mm x 4.6 mm,5 m), Hypurity C18 (50mm x 4.6 mm, 5 m) and Rypurity Advance (50 mm x 4.0 mm, 5 m), YMC basic (50 mm x2 mm, 5 m), Zorbax Eclipse Plus C18, (2.1mm x 50 mm, 3.5 µm) were tried during the method development. The best signal and good peak shape was obtained using the Zorbax Eclipse Plus C18, 2.1 x 50 mm, 3.5mm, column. It gave satisfactory peak shapes for both Omadacycline and Omadacycline-D9. Flow rate of 0.3mL/min without splitter as used and reduced the run time to 3.0 min. Both Drug and Internal Standard were eluted with shorter time at 2.0 min. For an LC-MS/MS analysis, utilization of stable isotope- labeled or suitable analog drugs as an internal standard proves helpful when a significant matrix effect is possible. In our case, Omadacycline-D9 was found to be best for the present purpose. The column oven temperature was kept at a constant temperature of about 45°C.
[0016] Injection volume of 10lL sample is adjusted for better ionization and chromatography. Prior to load the sample for LC injection, the co-extracted proteins should be removed from the prepared solution. For this purpose, initially we tested with different extraction procedures like PPT (Protein Precipitation), LLE (Liquid Liquid extraction), and SPE (Solid Phase extraction). We found ion suppression effect in protein precipitation method for drug and internal standard. Further, we tried with SPE and LLE. Out of all, we observed LLE is suitable for extraction of drug and IS. We tried with several organic solvents (ethyl acetate, chloroform, n-hexane, dichloro methane and methyl tertiary butyl ether) individually as well with combinations in LLE to extract analyte from the plasma sample. In our case methyl tertiary butyl ether: dichloromethane (75:25) combination served as good extraction solvent. Autosampler wash is optimized as 80% methanol. Several compounds were investigated to find a suitable IS, and finally Omadacycline-D9 found the most appropriate internal standard for the present purpose. There was no significant effect of IS on analyte recovery, sensitivity or ion suppression. Righ recovery and selectivity was observed in the Liquid-Liquid extraction method. These optimized detection parameters, chromatographic conditions and extraction procedure resulted in reduced analysis time with accurate and precise detection of Omadacycline in human plasma
[0017] Chromatographic conditions are obtained by Zorbax Eclipse Plus C18, 2.1 x 50 mm, 3.5 µm, was selected as the analytical column. Column temperature was set at 45°C. Mobile phase composition was 0.1% formic acid: methanol (80:20 vlv). Source flow rate 300 µL/min without split. Injection volume of 10 µL. Omadacycline and Omadacycline -D9 mesylate were eluted at 1.2 ± 0.2 min, with a total run time of 3.0 min for each sample
[0018] FIG. 2 illustrates a exemplary view of validating the specific RPLC-MS method for the determination of omadacycline in human plasma samples accordance with embodiments of the present disclosure. Liquid-Liquid extraction procedure was used for isolation of Omadacycline from the plasma samples. For this purpose, 50µL of Omadacycline -D9 (IS) concentration of 10nglmL) 100µL plasma (respective concentration of plasma sample) was added into ria vials then vortexed approximately. Followed by 200 µl of 1M Na2C03 solution, 3mL of Extraction solvent (MTBE: DCM (3:1, vlv) was added to each tube and vortexed for 10 minutes. After that, the samples were centrifuged at 4000 rpm for approximately 10 minutes at 20°C temperature and transfer the supernatant into respective ria vials. These samples were allowed to evaporate until dryness under nitrogen stream at 25°C. Finally, the residue was reconstituted with 200µL of reconstitution solution (MeOH: 0.1% formic acid (1:4). Further samples were centrifuged at 4000 rpm for approximately 2 minutes and at 20°C and supernatant were transferred into auto sampler vials with caps and 10 1L of sample was injected onto the LC-MS/MS system
[0019] Calibration curve parameters and regression model includes the analytical curves of Omadacycline were constructed in the concentrations ranging from 5.0 -12000.0 pglmL in human plasma. Calibration curves were obtained by weighted linear regression (weighing factor: 1/x2). The ratio of Omadacycline peak area to Omadacycline-D9 peak area was plotted against the ratio of Omadacyclinegiline concentration in nglmL. The fitness of calibration curve was confirmed by back-calculating the concentrations of calibration standards.
[0020] The method development conclusion is obtained by the developed method is suitable for estimation of plasma concentrations of Omadacycline as a single analytical run, in plasma samples from Bioequivalence and Pharmacokinetic studies. This was followed by method validation. The objective of the bioanalytical method validation is to validate specific rplc-ms method for the determination of omadacycline in human plasma samples.
[0021] Representative chromatograms of Plasma blank, blank +IS, LOQ, VLOQ, LLOQ, LQC, MQC, RQC samples, Calibration curve .
[0022] Bioanalytical Method Validation Parameters is obtained by system suitability. The system suitability includes a six replicate injections of aqueous mid QC along with internal standard were injected and the results were depicted.

System suitability of Omadacycline (OM) and Omadacycline-D9 (OMD9)
Injection number Peak area
Analyte (OM) Internal standard
(OMD9)
1 5543220 457843
2 5743214 467841
3 5743674 457843
4 5643240 457140
5 5743282 477842
6 5743280 457843
N 5693318 462725
Mean 83732 8454
SD (±) 1.47 1.83
% CV (%RSD) 5543220 457843
[0023] The acceptance criteria is %CV (%RSD) for the analyte and internal standard should not be more than 5%.
[0024] The Selectivity (Matrix Screening) and Specificity includes ten lots of blank human plasma samples were analyzed out of which six lots free from interference were selected for assessing the selectivity, specificity and the results were depicted.
[0025] The Acceptance criteria is Endogenous/potential interfering peak areas for blank samples must be less than 20% of the LLOQ peak area of analyte retention time and less than 5% for IS retention time.
Plasma B. No LLOQ Peak area LLOQ S/N ratio
(Lot.No-1) 3481 14
3858 12.6
3923 12.7
3935 12.6
3965 14.9
4864 18.4
N 4004 14.2
Mean 457.67 2.26
[0026] The Acceptance criteria is the mean S/N ratio of LLOQ should be ≥ 5.
[0027] An injector Carry Over includes an injector carryover was assessed by injecting the extracted blank samples followed by extracted ULOQ, LLOQ samples and %carry over was calculated. The results were depicted.


Table7: Injector carryover of AT and ATD8
Injection No Sample Analyte (OM) Internal standard (OMD9)
Peak area %carry over Peak area %carry over
response response
i. Extracted blank 0 0 0 0
ii. Extracted LLOQ 56731 43245
iii. Extracted ULOQ 8342231 43426
iv. Extracted blank 0 0

[0028] The Acceptance criteria is interfering peak areas for blank samples must be less than 20% of the LLOQ peak area of analyte retention time and less than 5% for IS retention time.
[0029] The Linearity (Calibration Curve) includes a Calibration standards were prepared to obtain linearity range of 5.00 to 12000pg/mL and assayed in five replicates on five different days and the results were depicted in Table 8.

Analytical Nominal Concentration (pg/mL)
Run ID CS1 CS2 CS3 CS4 CS5 CS6
(5.00 pg/mL) (10.00 pg/mL) (100.00 pg/mL) (600.00 pg/mL) (1200.00 pg/mL) (2400.00 pg/mL)
P&A Batch-1 4.9 9.9 98.3 597.4 1204.8 2469.6
P&A Batch-2 5 9.66 96.6 599.6 1189.2 2318.4
P&A Batch-3 4.9 9.03 90.3 602.8 1183.6 2467.2
P&A Batch-4 5.1 9.82 98.2 599.2 1158.4 2386.8
P&A Batch-5 4.9 9.45 94.5 600.4 1184 2398
N 5 5 5 5 5 5
Mean 4.96 8.81 95.58 599.88 1184 2408
SD(±) 0.09 0.35 3.33 1.97 16.7 63
CV% 1.8 3.95 3.48 0.33 1.41 2.62
Mean %Accuracy 99.2 88.1 95.58 99.98 98.67 100.33

Analytical run ID
CS7
(4800.00 pg/mL) CS8
(7200.00 pg/mL) CS9
(9600.00 pg/mL) CS10
(12000.00 pg/mL)
P&A Batch-1 4739.2 7208.8 9178.4 12071.5
P&A Batch-2 4636.8 6955.2 9273.6 11907.9
P&A Batch-3 4734.4 6501.6 8768.8 11944.4
P&A Batch-4 4433.6 6950.4 8867.2 11689.8
P&A Batch-5 4836.0 6904.0 9072.0 11853.4
N 5 5 5 5
Mean 4676.00 6904.00 9032.00 11893.40
SD(±) 152.72 254.76 210.85 139.27
CV% 3.27 3.69 2.33 1.17
Mean %Accuracy 97.42 95.89 94.08 99.11
[0030] Mean %accuracy for LLOQ (CS1) should be 100±20% and for CS2 to CS11 should be 100±15%. The % CV should ≤ 15%, except lowest calibration standard (CS1) for which it is ≤ 20% and correlation coefficient should be greater than (r2) 0.9980.
[0031] One set of calibration standards (CS1 to CS11) and one set contains four different concentrations of quality control standards of Lower limit QC (5.00 pg/mL), Low QC (15.00 pg/mL), Mid QC (4500.00 pg/mL) and High QC (9000.00 pg/mL) concentrations were prepared in screened human plasma and analyzed each quality control (QC) standards in six replicates on the same day. The results were depicted in Table 9.

P&A LLOQ Low QC Mid QC High QC
Batch ID (5.00 pg/mL) (15.00 pg/mL) (4500.00 pg/mL) (9000.00 pg/mL)
Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy
P&A 5.1 102 14.9 99.3 4450.6 98.9 9012.5 100.1
Batch 1 4.8 96 15.2 101.3 4612.4 102.5 8976.6 99.7
4.7 94 14.8 98.7 4521.3 100.5 8956.8 99.5
4.8 96 14.7 98 4531.2 100.7 9876.4 109.7
5.2 104 14.6 97.3 4456.5 99 8897.5 98.9
4.8 96 15.3 102 4412.7 98.1 9056.7 100.6
N 6 6 6 6
Mean 4.79 14.92 4497.45 9129.42
SD (±) 0.16 0.23 65.87 411.56
CV (%) 3.42 1.54 1.46 4.51
Mean% Accuracy 95.83 99.44 99.94 101.44
[0032] Precision (% CV) should be ≤ 15% for LQC, MQC and HQC quality control standards, except LLOQ for which it is ≤ 20%. Mean % Accuracy should be 100±15%, except LLOQ for which it is 100±20%.
[0033] Inter batch Precision and Accuracy/ Intermediate Precision / Ruggedness / Reproducibility / Under Different Conditions / Between batch includes an o ne set of calibration standards (CS1 to CS11) and one set contains four different concentrations of quality control standards of Lower limit QC (5.00 pg/mL), Low QC (15.00 pg/mL), Mid QC (4500.00 pg/mL) and High QC (9000.00 pg/mL) concentrations were prepared in screened human plasma and analyzed each quality control (QC) standards in six replicates on five different days. The results were depicted in Table 10.


P&A LLOQ Low QC Mid QC High QC
Batch ID (5.00 pg/mL) (15.00 pg/mL) (4500.00 pg/mL) (9000.00 pg/mL)
Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy
P&A 5.1 102 14.9 99.33 4450.6 98.9 9012.5 100.14
Batch 1 4.8 96 15.2 101.33 4612.4 102.5 8976.6 99.74
(Day-1) 4.7 94 14.8 98.67 4521.3 100.47 8956.8 99.52
4.8 96 14.7 98 4531.2 100.69 9876.4 109.74
5.2 104 14.6 97.33 4456.5 99.03 8897.5 98.86
4.6 92 14.1 94 4451.5 98.92 8899.5 98.88
P&A 4.75 94.93 15.1 100.67 4220.6 93.79 9114.95 101.28
Batch 2 4.71 94.25 14.5 96.67 4572.4 101.61 9118.3 101.31
(Day-2) 4.68 93.56 14.2 94.67 4545.2 101 9121.65 101.35
4.64 92.88 14.8 98.67 4678.5 103.97 9125 101.39
4.61 92.19 15.2 101.33 4478.2 99.52 9128.35 101.43
4.58 91.5 14.64 97.59 4519.16 100.43 9131.7 101.46
P&A 4.54 90.82 14.62 97.48 4522.07 100.49 9135.06 101.5
Batch 3 4.51 90.13 14.61 97.37 4524.99 100.56 9138.41 101.54
(Day-3) 4.47 89.45 14.59 97.26 4527.9 100.62 9141.76 101.58
4.44 88.76 14.57 97.15 4530.82 100.68 9145.11 101.61
4.4 88.08 14.56 97.04 4533.73 100.75 9148.46 101.65
4.37 87.39 14.54 96.93 4536.65 100.81 9151.81 101.69
P&A 4.34 86.7 14.52 96.82 4539.56 100.88 9155.16 101.72
Batch 4
(Day-4) 4.3 86.02 14.51 96.72 4542.48 100.94 9158.52 101.76
[0034] Acceptance criteria includes Precision (%CV) should be ≤ 15% for LQC, MQC and HQC quality control standards, except LLOQ for which it is ≤ 20%. Mean % Accuracy should be 100±15%, except LLOQ for which it is 100±20%.
[0035] The Extraction efficiency (Recovery) includes the extraction recovery was determined in sextuplicate by comparing the extracted QC standards with un-extracted QC standards at three different concentrations of low (15.00 pg/mL), medium (4500.00 pg/mL), high (9000.00 pg/mL) and the results were depicted in
[0036] Table 11.
[0037]
[0038]
Standard
conc (pg/mL) Unextracted OM
peak area Extracted OM
peak area Unextracted
OMD9
peak area Extracted
OMD9
peak area
Low QC
(15.00 pg/mL) 765 720 47921 46993
758 720 47927 41485
787 721 47934 45674
742 743 46921 46876
765 743 46856 46981
732 741 47678 46323
N 6.00 6.00
Mean 731.33 45722.00
SD(±) 12.08 2137.66
% CV 1.65 4.68
% Recovery 94.12 98.06
Medium QC
(4500.00 pg/mL) 282875 276543 46999 46321
272657 265686 47078 42456
262543 267656 47221 46567
302653 270675 48437 46486
282789 279978 47890 46098
282321 279876 48967 46798
N 6 6
273402.33 45787.67
SD(±) 6245.19 1649.03
% CV 2.28 3.60
% Recovery 97.76 98.56
High QC
(9000.0 pg/mL) 507658 487654 47543 46769
507653 497657 47231 46786
507628 489876 47543 44643
507616 489876 46789 46935
507601 488312 47156 45679
507586 478212 46984 46568
N 6 6 6
Mean 488597.83 46230.00
SD(±) 6231.76 898.12
% CV 1.28 1.94
% Recovery 96.06 98.37
Mean
%Recovery 94.12 91.68
Mean
% CV 5.12 7.09
[0039] Acceptance criteria includes the mean coefficient of variation for LQC, MQC and HQC shall not exceed 25%. The mean coefficient of variation for IS shall not exceed 25%.
[0040] Matrix Effect includes a six extracted blank plasma samples in three replicates spiked with the un-extracted concentration of mid QC (3000.00 pg/mL) were compared with un-extracted standards of the same concentration and the results were depicted in Table 12.


Blank Plasma
Lot.No Blank Plasma
B.No MQC (4500.00 pg/mL)
Extracted
Peak area ratio Unextracted Peak area ratio Matrix
factor
1 22-10342 A1
0.11 0.11 1.00
0.12 0.12 1.00
0.11 0.12 0.92
2
22-10342 A2
0.11 0.12 0.92
0.14 0.14 1.00
0.14 0.13 1.08
3
22-10342 A3 0.11 0.11 1.00
0.12 0.12 1.00
0.11 0.12 0.92
4
22-10342 A4
0.13 0.13 1.00
0.12 0.14 0.89
0.14 0.14 1.00
5
22-10342 A 5
0.11 0.11 1.00
0.12 0.12 1.00
0.12 0.11 1.09
6
22-10342 A6 0.13 0.13 1.00
0.12 0.14 0.89
0.14 0.14 1.00
N 18 18 18
Grand Mean 0.98
SD(±) 0.06
CV (%) 5.84
[0041] Acceptance criteria includes the precision of the matrix factor should be less than %CV ≤ 15%.
[0042] Dilution integrity includes a dilution integrity was carried in sextuplicate by 1:1 (DQC1) dilution and 1:4 dilution (DQC2) of 1.5 × ULOQ standard with screened blank rat plasma, whereas DQC1 and DQC2 standards were treated as un-known samples and calculated concentration was found from the standard curve regression equation.
[0043] P&A
[0044] Batch ID DQC1
Dilution factor: ½
Nominal conc: 9000.00 pg/mL DQC2
Dilution factor: ¼
Nominal conc: 4500.00 pg/mL
Conc. Found %Accuracy Conc. Found %Accuracy
P&A
Batch 5 9001 100.01 4511 100.24
9003 100.03 4501 100.02
9022 100.24 4512 100.27
9013 100.14 4501 100.02
9004 100.04 4504 100.09
9001 100.01 4502 100.04
N 6 6
Mean 9007.33 4505.17
SD (±) 8.45 5.04
CV (%) 0.09 0.11
Mean
%Accuracy 100.08 100.11

[0045] Acceptance criteria includes the % CV should be ≤ 15% for both dilutions (DQC1 and DQC2) and the mean %accuracy was within ±15% of the actual value DQC1 and DQC2 standards.
[0046] Re-Injection stability includes the re-injection stability of OM, QC samples (LQC and HQC) of P&A batch-2 samples (Pre-analyzed samples) were set aside on the bench at room temperature up to 26 h after the initial analysis. Stability samples (RI stability samples) were re-injected after 26 h to determine P&A of the method.
[0047]
P&A
Batch ID Low QC (15.00 pg/mL) High QC (9000.00 pg/mL)
Pre-analyzed
sample conc.
(pg/mL) RI stability sample conc.
(pg/mL) Pre-analyzed
sample conc.
(pg/mL) RI stability sample conc.
(pg/mL)
P&A
Batch 2 13.69 13.79 8573.46 8342.68
15.21 13.78 8725.71 8076.75
15.09 14.23 8716.07 8683.61
14.54 18.7 8694.75 8281.71
14.41 14.1 9070.18 8292.64
15.48 15.3 8874.75 8333.36
N 6 6 6 6
Mean 14.74 14.98 8775.82 8335.13
SD(±) 0.65 1.90 173.23 196.43
%CV 4.44 12.71 1.97 2.36
Mean
% Accuracy 101.79 99.89 102.55 107.98
[0048] Acceptance criteria includes the % CV should be ≤ 15% Except LLOQ for which it is ≤ 20% and mean % accuracy should be 100±15%.
[0049] Ruggedness with Different Analyst includes a standards of P&A batch-3 and sextuplets of Lower limit QC, lower QC, Mid QC, high QC was processed by different analyst.
P&A
Batch ID LLOQ
(5.00 pg/mL) Low QC
(15.00 pg/mL) Mid QC
(4500.00 pg/mL) High QC
(9000.00 pg/mL)
Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2 Analyst ID 1 Analyst ID 2
P&A
Batch 3 5.11 5.15 15.19 15.06 4500.19 4500.13 9000.24 9007.10
5.10 5.16 15.18 15.07 4500.90 4500.90 9001.93 9000.21
5.12 5.22 15.05 15.07 4507.17 4500.11 9000.22 9000.30
5.21 5.14 15.07 15.72 4500.12 4500.12 9006.22 9000.10
5.12 5.11 15.04 15.04 4521.19 4500.16 9000.18 9000.19
5.14 5.17 15.05 15.06 4503.93 4500.66 9003.50 9009.90
N 6 6 6 6 6 6 6 6
Mean 5.13 5.16 15.10 15.17 4505.58 4500.35 9002.05 9002.97
SD (±) 0.04 0.04 0.07 0.27 8.12 0.34 2.43 4.38
CV (%) 0.78 0.71 0.46 1.78 0.18 0.01 0.03 0.05
Mean
%Accuracy 102.67 103.17 99.36 98.88 100.12 99.99 99.98 99.97

[0050] Acceptance criteria includes the % CV should be ≤ 15 %, except LLOQ for which it is ≤ 20% and the mean % accuracy should be 100±15%, except LLOQ for which it is 100±20%.
[0051] The Ruggedness with Different Column includes a standards of P&A batch-5 and sextuplets of LLOQ, LQC, MQC, HQC were re-injected processed by different columns of varying the batch number with same column specifications. The results were depicted in Table 16
P&A
Batch ID LLOQ
(5.00 pg/mL) Low QC
(15.00 pg/mL) Mid QC
(4500.00 pg/mL) High QC
(9000.00 pg/mL)
Column ID LC/134 Column ID LC/145 Column ID LC/134 Column ID LC/145 Column ID LC/134 Column ID LC/145 Column ID LC/134 Column ID LC/145
P&A
Batch 5 5.12 5.15 15.12 15.06 4500.13 4500.11 9000.24 9000.10
5.10 5.16 15.11 15.03 4500.19 4500.90 9000.20 9000.29
5.12 5.17 15.05 15.06 4500.13 4500.11 9000.40 9000.39
5.22 5.15 15.05 15.11 4500.10 4500.14 9000.22 9000.15
5.12 5.11 15.04 15.04 4500.14 4500.16 9000.11 9000.19
5.14 5.17 15.05 15.06 4500.13 4500.51 9000.21 9000.90
N 6 6 6 6 6 6 6 6
Mean 5.14 5.15 15.07 15.06 4500.14 4500.32 9000.23 9000.34
SD(±) 0.04 0.02 0.04 0.03 0.03 0.32 0.09 0.29
CV (%) 0.83 0.43 0.23 0.18 0.00 0.01 0.00 0.00
Mean
%Accuracy 102.73 103.03 99.54 99.60 100.00 99.99 100.00 100.00
[0052] Acceptance criteria includes the % CV ≤ 15 %, except LLOQ for which it is ≤ 20% and the mean % accuracy should be 100±15%, except LLOQ for which it is 100±20%.
[0053] The Bench top Stability / Room Temperature Stability (Room temp, 24 h) includes six replicates of spiked low and high concentrations (OM stability samples) were set aside at ambient temperature up to 24 h. Samples were processed and compared with newly prepared low and high concentrations (comparison samples).
P&A
Batch ID Low QC (15.00 pg/mL) High QC ( 9000.00 pg/mL)
Comparison samples
(0 h) OM Stability samples
(24 h) Comparison samples
(0 h) OM Stability samples
(24 h)
Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy
P&A
Batch 6 15.96 106.40 14.13 94.20 8846.25 98.29 10178.68 113.10
15.56 103.73 15.1 100.67 8906.46 98.96 9023.68 100.26
14.99 99.93 15.03 100.20 9977.25 110.86 9378.75 104.21
15.73 104.87 16.09 107.27 9252 102.80 9777.11 108.63
15.3 102.00 15.48 103.20 8920.71 99.12 9416.57 104.63
16.26 108.40 14.13 94.20 9105.64 101.17 9656.36 107.29
N 6.00 6.00 6.00 6.00
Mean 15.63 14.99 9168.05 9571.86
SD(±) 0.46 0.77 424.01 394.82
CV (%) 2.92 5.12 4.62 4.12
[0054] Acceptance criteria includes the % CV ≤ 15 %, except LLOQ for which it is ≤ 20% and the mean %accuracy should be 100±15%.
[0055] Freeze and thaw stability (after 3rd cycle at -30°C) includes a six replicates of low and high concentrations (FT stability samples) were frozen at -30°C and subjected to three freeze-thaw cycles of 24, 36 and 48 h (-30°C to room temperature) and compared with newly prepared low and high concentrations (comparison samples).
[0056] P&A
[0057] Batch ID Low QC (15.00 pg/mL) High QC ( 9000.00 pg/mL)
Comparison samples FT Stability sample
at 4th cycle Comparison samples FT Stability sample
at 4th cycle
Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy
P&A
Batch 6 15.96 106.40 16.59 90.42 8846.25 98.29 9784.29 91.98
15.56 103.73 16.24 108.27 8906.46 98.96 10030.71 111.45
14.99 99.93 16.84 89.07 9977.25 90.21 8677.39 103.72
15.73 104.87 15.53 103.53 9252 102.80 9145.82 101.62
15.3 102.00 15.91 106.07 8920.71 99.12 9916.29 110.18
16.26 108.40 16.06 99.38 9105.64 102.93 9364.07 94.47
N 6.00 6.00 6.00 6.00
Mean 15.63 16.20 9168.05 9486.43
SD(±) 0.46 0.47 424.01 520.63
CV (%) 2.92 2.92 4.62 5.49
Mean
%Accuracy 104.22 107.97 101.87 94.87

[0058] Acceptance criteria includes the % CV ≤ 15 %, except LLOQ for which it is ≤ 20% and mean %accuracy should be 100±15%. Six replicates of low and high concentrations (AS stability samples) were stored in auto-sampler up to 65 h at 2-8°C. Stability samples were compared with newly prepared low and high concentrations (comparison samples).
[0059] P&A
[0060] Batch ID Low QC (15.00 pg/mL) High QC ( 9000.00 pg/mL)
Comparison samples (0 h) AS Stability samples (65 h) Comparison samples (0 h) AS Stability samples (65 h)
Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy Conc. found % Accuracy
P&A
Batch 6 13.69 91.25 14.83 98.83 8573.46 95.26 8343.64 92.71
15.21 101.42 14.36 95.75 8725.71 96.95 8662.29 96.25
15.09 100.58 14.86 99.08 8716.07 96.85 8444.25 93.83
14.54 96.92 15.28 101.83 8694.75 96.61 8196.21 91.07
14.41 96.08 14.76 98.42 9070.18 100.78 8873.79 98.60
15.48 103.17 14.66 97.75 8874.75 98.61 8793.21 97.70
N 6
6
6
6
Mean 14.74 14.79 8775.82 8552.23
SD(±) 0.65 0.30 173.23 266.65
CV (%) 4.44 2.01 1.97 3.12
Mean
%Accuracy
98.24 98.61 97.51 95.02

[0061] Acceptance criteria includes a mean %accuracy was 85-115% and % CV ≤ 15%.
[0062] Long-term Stability (-30°C, 64 Days) includes after completion of the stability period stored at -30 °C (45 days) six replicates of low and high concentrations (LT stability samples) were compared with newly prepared low and high concentrations (comparison samples).
P&A
Batch ID Low QC (15.00 pg/mL) High QC ( 9000.00 pg/mL)
Comparison samples (0 h) LT Stability samples
(64 days) Comparison samples (0 h) LT Stability samples (64 days)
Conc. found % nominal Conc. found % nominal Conc. found % nominal Conc. found % nominal
P&A
Batch 6 14.84 98.92 15.63 104.17 8455.82 93.95 8576.14 95.29
14.26 95.08 14.80 98.67 8786.36 97.63 8458.50 93.98
14.10 94.00 15.16 101.08 9116.68 101.30 8428.82 93.65
14.28 95.17 15.49 103.25 9141.32 101.57 8921.79 99.13
14.06 93.75 15.98 106.50 9232.29 102.58 8823.32 98.04
15.00 100.00 15.68 104.50 8573.04 95.26 8267.57 91.86
N 6 6 6 6
[0063] Acceptance criteria includes mean % accuracy should be 85-115 % and % CV should be ≤ 15%. Short-term Stock Solution Stability of Omadacyclin, OmadacyclinD9 at Room Temperature and Spiking Solution (Working Conc.) of Internal Standard (OmadacyclinD9) at Refrigerated Conditions (2-8°C) includes room temperature stock solution stability was determined using standard stock solutions of OM and OMD9 (ST stability samples) were set aside on the bench up to 9.5 h and compared with newly prepared stock solutions (comparison samples).
[0064] Refrigerated IS (OMD9) spiking solution stability (IS stability samples) was determined using IS spiking solution of OMD9 was set aside in refrigerator (2-8°C) up to ten days and compared newly prepared IS stock solution samples (comparison samples).
[0065] Omacycline OmadacyclineD9
Comparison samples
peak area
(0 h) ST Stability samples
peak area
(9 h) Comparison samples
peak area
(0 h) ST Stability samples
peak area
(9.5 h)
567282 580642 137431 138144
585470 579028 139566 139479
565590 576135 139400 139391
576170 574545 136221 143864
568253 580468 140427 138147
578242 577151 137731 142314
N 6 6 6 6
Mean 573501.17 577994.83 138462.67 140223.17
SD (±) 7767.35 2460.24 1586.15 2345.50
CV (%) 1.35 0.43 1.15 1.67
Mean %Accuracy 101.10 101.51

Comparison samples
peak area
(0 h) ST Stability samples
peak area
(10 days at refrigerated conditions)
140514 141485
138527 141777
142024 141138
138838 142939
144045 138636
136663 139908
N 6 6
Mean 140101.83 140980.50
SD (±) 2657.67 1509.73
CV (%) 1.90 1.07
Mean
% Accuracy 99.64
[0066] Acceptance criteria: Mean %Accuracy should be 85-115 % and % CV should ≤ 15%. Method Validation Conclusion Omadacycline and Omadacycline-D9 includes that the validated method was characterized with an adequate rapid, exact, specific, responsive, rugged and steady.
[0067] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.