INTRODUCTION:

Decitabine¹ is chemically 4-amino-1-(2-deoxy-β-D-ERYTHRO-PENTOFURANOSYL)-1, 3, 5-triazin-2(1H)-one and molecular formula is C₁₈H₁₂N₄O₄ and molecular weight is 228.21 g/mol. Decitabine a hypomethylating agent. Decitabine is believed to exert its antineoplastic² effects after phosphorylation and direct incorporation into DNA and inhibition of DNA methyltransferase, causing hypomethylation of DNA and cellular differentiation or apoptosis. It hypomethylates DNA by inhibiting the methyl transferase enzyme. It is useful to treatment of patients with myelodysplastic syndrome (MDS). The chemical structure of Decitabine as shown in Fig.1.

Fig. 1 Chemical structure of Decitabine

Literature survey revealed that a few analytical methods have been established for the estimation of decitabine^{3, 4, 5}. To the best of our knowledge, there is no reported HPLC method for estimation of decitabine related substances in drug dosage forms. The aim and objective of this study was to develop and validate the related substances of Decitabine in Decitabine lyophilized injection 50mg/vial.

MATERIALS AND METHODS:

Chemicals and reagents:

Decitabine standard was a generous gift from a reputed pharmaceutical organization. Decitabine lyophilized for injection 50mg/Vial (Natco, India) was purchased from local market. Gradient grade Acetonitrile was procured from Merck, India. Ammonium acetate and DMSO were purchased from Loba Chemie, Mumbai, India. Water used in the HPLC analysis was prepared by the water purifier Arium, 611UF, Sartorius, Germany. The mobile phase filtered through a 0.45µm membrane filter, fisher brand and all the test solutions were filtered through a 0.45 μm millex PVDF filter, Millipore, India.

The method has been satisfactorily applied to the simultaneous estimation of related substances of Decitabine in bulk and pharmaceutical dosage forms like injection and tablets form.

Method development and optimization:

A simple reverse phase-HPLC method for the analysis of Decitabine related substances in pharmaceutical dosage form. In method development⁶ the solubility of the active pharmaceutical ingredient was checked in different solvents like water, methanol and acetonitrile. The decitabine is soluble in DMSO at 90mg/mL, soluble in Ethanol at 2mg/mL with warming and soluble in water at 25mg/mL with warming. Finally the standard and samples are diluted by Dimethyl sulfoxide.

The different mobile phases like phosphate buffer and methanol were used in various compositions with isocratic pump mode a flow rate of 1.0 mL/min but impurities peaks not separated and finally by changing the buffer, mobile phase, pump mode and flow rate.

The chromatographic analysis was tested by using different columns like waters Exterra C18, 250 X 4.6mm, 5µm particle size columns maintained at different temperatures 40°C, 45°C and 50°C were used, but the impurities separation was not achieved. The actual chromatography analysis achieved on Inertsil ODS-3V, 250 x 4.6 mm, 5µm particle size by using ammonium acetate buffer is used as a mobile phase-A and water, acetonitrile in the ratio of 10:90 %v/v used as mobile phase-B with gradient elution.

Method:

Preparation of Ammonium acetate buffer:

Weigh and transfer 1.54 g of Ammonium acetate in 1 Liter of water sonicate to dissolve and filter the solution through 0.45 µm membrane filter and degas.

Mobile phase Preparation:

Mobile phase A:

Use ammonium acetate buffer as mobile phase solution A.

Mobile phase B:

Water: Acetonitrile (10:90 %v/v)

Diluent:

Use Dimethyl sulfoxide as diluent.

Preparation of Decitabine standard stock solution (250ppm):

Take and transfer about 12.5 mg of Decitabine working standard into a 50 mL volumetric flask, add 10mL of diluent sonicate to dissolve and dilute to volume with diluent and mix well.

Preparation of Decitabine standard solution (2.5ppm):

Pipette 1 mL of Decitabine standard stock solution in to 100 mL volumetric flask and dilute to volume with diluent and mix well.

Preparation of Sample solution (2500 ppm):

Take and transfer about 50 mg of Decitabine drug substances into a 20 mL volumetric flask, add 10 mL of diluent sonicate to dissolve and dilute to volume with diluent and mix well.

Placebo solution:

Take and transfer placebo powder equivalent to 50 mg of Decitabine into a 20 mL of volumetric flask, add 10 mL of diluent sonicate to dissolve and dilute to volume with diluent and mix well.

Chromatographic conditions:

Column : Inertsil ODS-3V, 250 x 4.6 mm, 5µm

Flow rate : 1.2 mL/Min

Injection volume : 10.0 µL

Column temperature : 35° C

Auto sampler temperature : 25° C

Wavelength : 254nm

Run time : 20 Minutes for standard and 70 Minutes for Blank, Placebo and samples

Elution Mode : Gradient

Note: (Refer Table1 for standard and Table2 for Blank, Placebo and samples)

Table 1: Gradient Program

Time (Minutes)	Flow	Mobile phase-A	Mobile phase-B	Curve
Initial	1.2	100	0	Initial
20.0	1.2	100	0	6

Table 2: Gradient Program

Time (Minutes)	Flow	Mobile phase-A	Mobile phase-B	Curve
Initial	1.2	100	0	Initial
20.0	1.2	100	0	6
25.0	1.2	80	20	6
40.0	1.2	35	65	6
45.0	1.2	35	65	6
50.0	1.2	10	90	6
55.0	1.2	10	90	6
60.0	1.2	100	0	6
70.0	1.2	100	0	6

Method Validation:

The method was validated in accordance with recognized ICH guidelines^{7, 8}.

System suitability:

Prepared the standard solution as per methodology and injected six times into the chromatographic system and obtained % RSD from six replicate injections was 0.7. The observed tailing factor for Decitabine peak from the first injection of standard solution was 1.0 and system suitability results are given in Table 3.

Table 3: System suitability results

Injection#	Peak area of Decitabine
1	30660
2	30751
3	30310
4	30358
5	30290
6	30357
Mean	30454
SD	198.36
%RSD	0.7

Specificity:

Specificity is demonstrated by checking the blank, placebo, known and degradant impurities interference with the analyte peak.

Prepared blank, placebo, standard solution, sample solution, Impurities spiked sample solution and individual impurities solutions as per method and injected into HPLC system to evaluate the peak purity and interference of any peak with Decitabine and known impurities.

All blank and placebo peaks were well separated from known impurities and Decitabine peak. All known and degradant peaks were separated with each other and Decitabine peak. Specificity results are addressed in table 4.

Table 4: Specificity results

Name of the Active/Impurity	Retention time (min)	Peak purity
Name of the Active/Impurity	Retention time (min)	Purity angle	Purity threshold
α- Decitabine(α-SPT1223API)	9.166	0.254	0.341
α-isomer (SPT1223M2)	44.174	0.182	0.322
methyl 4-chlorobenzoate	45.484	0.211	0.444
β-isomer (SPT1223M2)	45.977	0.175	0.354
O-Acetyl(SPT1223M2)	54.548	0.128	0.324
Decitabine in spiked sample	11.853	0.116	0.222

Forced degradation:

Forced degradation of Decitabine in Decitabine injection was carried out, to confirm that, during the stability study or throughout the shelf life, any degradation product if found will not interfere with Decitabine and known impurities peaks and also the forced degradation study will help to identify the type of degradation pathway (whether oxidative, alkali hydrolysis, acid hydrolysis, photolytic, dry heat and humidity) for each of the degradants.

Decitabine injection was forcefully stressed by exposure to acid hydrolysis, base hydrolysis, peroxide, Hydrolysis, photo stability, Humidity and thermal. Control and stressed samples were injected into the HPLC system and evaluate the Peak purity, interference of degradants and mass balance.

In force degradation studies fortunately all generated impurities have not interfered with the Decitabine peak, known impurities peaks and also with each other. The purity angle of Decitabine and its known impurities is less than the purity threshold. The specificity chromatograms and peak purity plots are shown from fig.2 to fig.28. Results for forced degradation studies were addressed in the table 5 and table 6.

Blank, placebo and impurities have not shown any interference with Decitabine, Known impurities, all unknown and degradant impurities. Hence the above method is specific.

Fig.2: Chromatogram of Blank

Fig.3: Chromatogram of Placebo

Fig.4: Chromatogram of standard

Fig.5: Peak purity plot of Decitabine peak in standard

Fig.6: Chromatogram of Control sample

Fig.7: Peak purity plot of Decitabine peak in Control sample

Fig.8: Chromatogram of Spiked sample

Fig.9: Peak purity plot of Decitabine peak in spiked sample

Fig.10: Chromatogram of Alpha Decitabine impurity

Fig.11: Chromatogram of Alpha isomer

Figure.12: Chromatogram of 4-Chloro benzoate

Fig.13: Chromatogram of Beta Isomer

Fig.14: Chromatogram of O-Acetyl impurity

Fig.15: Chromatogram of Acid degradation sample

Fig.16: Peak purity plot of Decitabine peak in Acid degradation sample

Fig.17: Chromatogram of Base degradation sample

Fig.18: Peak purity plot of Decitabine peak in Base degradation sample

Fig.19: Chromatogram of Peroxide degradation sample

Fig.20: Peak purity plot of Decitabine peak in Peroxide degradation sample

Fig.21: Chromatogram of Hydrolysis degradation sample

Fig.22: Peak purity plot of Decitabine peak in Hydrolysis degradation sample

Fig.23: Chromatogram of Thermal degradation sample

Fig.24: Peak purity plot of Decitabine peak in Thermal degradation sample

Fig. 25: Chromatogram of Humidity degradation sample

Fig. 26: Peak purity plot of Decitabine peak in Humidity degradation sample

Fig. 27: Chromatogram of Photolytic degradation sample

Fig. 28: Peak purity plot of Decitabine peak in Photolytic degradation sample

Table 5: Forced degradation Studies

Sample Name	α- Decitabine (%w/w)	α-Isomer (%w/w)	β-Isomer (%w/w)	Methyl 4-chlorobenzoate (%w/w)	O-Acetyl (%w/w)	Imp at RRT 1.3(%w/w)
Control sample	ND	ND	ND	ND	ND	ND
0.01N HCl / 10min at Bench top and 10min at 40°C	0.273	ND	ND	ND	ND	6.834
0.01N NaOH / 10min at Bench top and 5min at 40°C	0.022	ND	ND	ND	ND	19.436
3.0%H₂O₂ for 10min on Bench top	ND	ND	ND	ND	ND	0.539
Water /40°C For 24Hrs	ND	ND	ND	ND	ND	0.268
Photolytic degradation (1.2million lux hours and 200 watt hr/m2)	ND	ND	ND	ND	ND	ND
Humidity 90%RH at 25°C for 5days	ND	ND	ND	ND	ND	4.159
Thermal 105°C for 6 Hours	ND	ND	ND	ND	ND	ND

Table 6:Forced degradation Studies

Sample Name	Single maximum unknown (%w/w)	Total Impurities (% w/w)	% of Assay	Mass Balance	Purity angle	Purity Threshold
Control sample	0.034	0.050	100.1	NA	0.230	15.447
0.01N HCl / 10min at Bench top and 10min at 40°C	3.963	12.649	92.7	97.1	0.413	22.405
0.01N NaOH / 10min at Bench top and 5min at 40°C	0.563	20.401	85.6	97.7	0.150	2.908
3.0%H₂O₂ for 10min on Bench top	0.0	0.539	98.3	98.7	0.176	25.952
Water /40°C For 24Hrs	0.0	0.268	100.0	100.1	0.251	9.665
Photolytic degradation (1.2million lux hours and 200 watt hr/m2)	0.051	0.116	99.2	99.2	0.321	8.699
Humidity 90%RH at 25°C for 5days	0.078	4.237	98.5	102.6	0.236	1.969
Thermal 105°C for 6 Hours	0.097	0.206	99.4	99.5	0.478	22.488

Table 8: Linearity of Decitabine and α- decitabine:

Linearity levels	Decitabine		α- decitabine
Linearity levels	Concentration (ppm)	Area response	Concentration (ppm)	Area response
LOQ	1.7142	11447	1.5812	10500
50 %	2.5712	16509	2.3718	14949
100 %	5.1425	32217	4.7435	28360
125 %	6.4281	40472	5.9294	35835
150 %	7.7137	47527	7.1153	42034
Correlation Coefficient (r)	1.000		1.000
Square Correlation Coefficient (r²)	0.999		0.999
Slope	6069.099		5740.629
Y-Intercept	1024.931		1373.867
Relative response factor	NA		0.95

Table 9: Linearity of α-isomer and Methyl 4-chlorobenzoate:

Linearity levels	α-isomer		Methyl 4-chlorobenzoate
Linearity levels	Concentration (ppm)	Area response		Concentration (ppm)
LOQ	2.4486	29290	LOQ	2.4486
50 %	3.6730	44317	50 %	3.6730
100 %	7.3459	86464	100 %	7.3459
125 %	9.1824	108623	125 %	9.1824
150 %	11.0189	127561	150 %	11.0189
Correlation Coefficient (r)	1.000		1.000
Square Correlation Coefficient (r²)	0.999		0.999
Slope	11519.67		8807.16
Y-Intercept	1680.179		811.795
Relative response factor	1.90		1.45

Table 10: Linearity of β-isomer and O-Acetyl

Linearity levels	β-isomer		O-Acetyl
Linearity levels	Concentration (ppm)	Area response	Concentration (ppm)	Concentration (ppm)
LOQ	2.4484	31626	LOQ	2.4484
50 %	3.6726	46973	50 %	3.6726
100 %	7.3452	91439	100 %	7.3452
125 %	9.1815	114926	125 %	9.1815
150 %	11.0178	134300	150 %	11.0178
Correlation Coefficient (r)	1.000		1.000
Square Correlation Coefficient (r²)	0.999		0.999
Slope	12077.79		8759.80
Y-Intercept	2531.743		1411.483
Relative response factor	1.99		1.44

Fig.29: Linearity graph for Decitabine

Fig.30: Linearity graph for α-Decitabine

Fig.31: Linearity graph for α-Isomer

Fig.32: Linearity graph for Methyl 4-Chlorobenzoate

Fig.33: Linearity graph for Methyl β-isomer

Fig.34: Linearity graph for O-Acetyl

Precision:

System precision:

It is demonstrated by calculating %RSD for retention time and peak areas of Decitabine peak from six replicate injections of standard solution preparation. The system precision results are addressed in table 11.

Table 11: System precision results

Injection#	Decitabine Retention time	Peak area of Decitabine
1	9.34	30660
2	9.34	30751
3	9.34	30310
4	9.35	30358
5	9.35	30290
6	9.34	30357
Mean	9.34	30454
SD	0.005	198.96
%RSD	0.1	0.7

Method precision / Intermediate precision:

Method precision was evaluated by injecting spiked known impurities on drug product at specification level. The results of Precision and Intermediate precision are reported from table 12 to table 14. % RSD values for both Precision study and Intermediate precision study are 4.1 for α- decitabine, 3.9 for α-isomer, 3.0 for Methyl 4-chlorobenzoate, 2.8 for β-isomer and 2.0 for O-Acetyl impurity. The data demonstrated that the values are met the acceptance criteria. Hence the method was found Precise.

Table 12: Method precision/Intermediate precision for α- decitabine and α-isomer (% Recovery)

Spiked sample	Method Precision	Intermediate Precision	Method Precision	Intermediate Precision
Spiked sample	α- decitabine (%)		α-isomer (%)
1	90.1	96.4	99.0	104.1
2	90.2	95.8	99.1	105.5
3	90.4	96.8	99.1	107.6
4	90.0	98.0	99.1	106.6
5	90.1	97.7	99.8	106.4
6	90.0	99.4	99.4	108.8
Mean	90.1	97.3	99.1	106.5
% RSD	0.2	1.3	0.2	1.5
Overall Mean (n=12)	93.7		102.8
Overall% RSD (n=12)	4.1		3.9

Table 13: Method precision / Intermediate precision for Methyl 4-chlorobenzoate and β-isomer (% Recovery):

Spiked sample	Method Precision	Intermediate Precision	Method Precision	Intermediate Precision
Spiked sample	Methyl 4-chlorobenzoate (%)		β-isomer (%)
1	95.3	98.3	97.0	97.8
2	95.4	96.9	97.1	98.1
3	94.8	98.3	96.8	100.4
4	94.3	101.4	96.8	102.6
5	93.6	100.4	96.2	102.3
6	93.4	101.0	96.5	104.1
Mean	94.5	99.4	96.7	100.9
% RSD	0.9	1.8	0.3	2.5
Overall Mean (n=12)	96.9		98.8
Overall% RSD (n=12)	3.0		2.8

Table 14: Method precision / Intermediate precision for O-Acetyl (% Recovery):

Spiked sample	Method Precision	Intermediate Precision
Spiked sample	O-Acetyl impurity (%)
1	99.4	102.8
2	99.8	102.9
3	99.4	103.3
4	100.1	103.4
5	99.3	102.9
6	99.7	105.0
Mean	99.6	103.4
% RSD	0.3	0.8
Overall Mean (n=12)	101.5
Overall% RSD(n=12)	2.0

Accuracy:

The accuracy was evaluated by measurement (n=3) applying the method to the sample spiked with known amounts of known impurities corresponding to LOQ, 50 %, 100 % and 150 % of specification. The recovery data of all known impurities obtained from a study of formulation from LOQ level to 150 %. The test sample were prepared at each % level and tested against standard according to the description of the methodology. The total average recovery for α- decitabine is 98.4 % with 0.5 % RSD, α-isomer is 102.7 % with 1.1 % RSD, Methyl 4-chlorobenzoate is 102.7 % with 0.8 % RSD, β-isomer is 101.4 % with 2.1 % RSD, O-Acetyl is 104.1 % with 0.4 % RSD. The accuracy results are addressed in table 15.

Based on the impurities recovery results, it is concluded that there was no interference from excipients present in the formulation and the method is accurate.

Name of the Active/Impurity	LOD (S/N Value)	LOD (Concentration in ppm)	LOQ (S/N Value)	LOQ (Concentration in ppm)
α- decitabine	3.4	0.4792	10.1	1.5812
α-isomer	3.8	0.7420	10.6	2.4486
Methyl 4-chlorobenzoate	3.1	0.7305	10.3	2.4103
β-isomer	3.6	0.7419	10.5	2.4484
O-Acetyl	3.2	0.7365	10.6	2.4304
Decitabine	3.6	0.5195	10.1	1.7142

At Room temperature (25°C) %Difference
Hours	Decitabine standard
Initial	NA
48 Hours	0.4
72 Hours	1.4

Parameter	Variation	Decitabine
Parameter	Variation	USP Tailing	% RSD from six replicate injections
Original conditions	None	1.0	1.3
Wavelength	251 nm	1.0	1.3
Wavelength	257 nm	1.3	3.8
Mobile phase variation	Organic 5% minus	1.0	0.6
Mobile phase variation	Organic 5% plus	1.0	0.5
Flow Rate mL/min	1.1mL/min	1.0	0.4
Flow Rate mL/min	1.3mL/min	1.1	0.5
Column oven temperature	30^oC	1.0	1.2
Column oven temperature	40^oC	1.0	1.0

RRT of Impurities in spiked sample
parameter	Variation	α-decitabine	α-isomer	Methyl 4-chlorobenzoate	β-isomer	O-Acetyl
Original conditions	None	0.78	3.58	3.70	3.74	4.43
Wavelength	251 nm	0.78	3.76	3.87	3.91	4.65
Wavelength	257 nm	0.78	3.76	3.87	3.91	4.65
Mobile phase variation	Organic 5% minus	0.79	3.70	3.80	3.84	4.58
Mobile phase variation	Organic 5% plus	0.79	3.83	3.94	4.01	4.75
Flow Rate mL/min	1.1mL/min	0.78	3.18	3.31	3.35	3.93
Flow Rate mL/min	1.3mL/min	0.78	3.64	3.74	3.78	4.51
Column oven temperature	30^oC	0.77	2.96	3.08	3.12	3.68
Column oven temperature	40^oC	0.80	3.97	4.09	4.14	4.90

Level	α-decitabine	α-isomer	Methyl 4-chlorobenzoate	β-isomer	O-Acetyl
LOQ Mean % Recovery	98.7	103.2	102.1	101.2	104.4
LOQ % RSD	0.8	0.2	0.3	0.2	0.3
50 % Mean % Recovery	98.0	103.5	103.3	103.6	103.9
50 % % RSD	0.2	1.9	1.4	3.5	0.3
100 % Mean % Recovery	98.4	102.3	102.8	100.6	104.2
100 % % RSD	0.4	0.3	0.2	0.1	0.3
150 % Mean % Recovery	98.3	101.8	102.6	100.0	103.7
150 % % RSD	0.2	0.9	0.7	1.0	0.2
Overall Mean % Recovery	98.4	102.7	102.7	101.4	104.1
Overall SD	0.48	1.16	0.85	2.17	0.37
%RSD Overall Recovery (%)	0.5	1.1	0.8	2.1	0.4