Assay of Doxepin Hydrochloride acc. to USP Monograph

Lee May May, Application Chemist

Abstract

This study describes a rugged high-performance liquid chromatography (HPLC) method for the assay of Doxepin Hydrochloride, following monograph guidelines as specified by the United States Pharmacopeia (USP). The method is proven to be highly stable with good reproducibility and excellent peak shapes using the fully porous particle (FPP) Purospher^® STAR RP-8e column over a wide concentration range of 0.01 to 1.0 mg/mL. The method complies with all USP specifications in terms of resolution, tailing factors, and RSD for both (E)- and (Z)-isomers.

Section Overview:

• Introduction
• Experimental
• Results
• Conclusion
• References

Introduction

Doxepin hydrochloride is a dibenzoxepin tricyclic compound used as an anti-depressant. The molecular formula of the compound is C₁₉H₂₁NO•HCl and its molecular weight is 315.84 Da. Doxepin HCl is made up of (E) and (Z) geometric isomers. It is a white crystalline powder freely soluble in water, methanol and dichloromethane.¹

The assay described below follows that of the USP monograph.² It uses the fully porous particle (FPP) Purospher^® STAR RP-8e column (USP code L7) for an extended doxepin concentration range from 0.01 to 1.0 mg/mL. 

Experimental

Table 1. LC conditions used for the doxepin hydrochloride determination

HPLC Parameters
Column:	Purospher® STAR RP-8 endcapped Hibar® RT 125 x 4 mm I.D., 5 µm (1.50033)
Mobile Phase:	[A] Buffer: 27.6 g/L monobasic sodium phosphate (NaH2PO4); [B] Methanol; A:B 70:30, adjusted to pH 2.5 with phosphoric acid; isocratic
Flow rate:	1 mL/min
Pressure Drop:	185 bar
Column temp.:	50 °C
Detector:	UV at 254 nm
Injection:	20 µL
Standard:	0.1 mg/mL USP Doxepin HCl RS in mobile phase

Results

System Suitability

USP monograph suitability requirements

• Run time: NLT 2 times the retention time of the (E) isomer
• Resolution: NLT 1.5 between the (E) and (Z) isomers.
• Tailing factor: NMT 2.0 for each (E) and (Z) isomers.
• Relative standard deviation: NMT 2.0% each of the (E)- and (z)-isomers

Acceptance criteria

• (E)-isomer: NLT 81.4% and NMT 88.2%.
• (Z)-isomer: NLT 13.6% and NMT 18.1%.

Table 2. Injection of 0.1 mg/mL doxepin HCl – Identification and system suitability

Peak No.	Analyte	RT (min)	Area (mAU*min)	% Peak area	Tailing factor	Resolution
1	(E)-isomer	22.55	31.917	84.93	1.00
2	(Z)-isomer	25.74	5.665	15.07	1.02	1.84
USP Requirement					NMT 2.0	NLT 1.5

Figure 1. Analysis of 0.1 mg/mL of doxepin HCl in mobile phase. The run time is NLT 2 times the retention time of the (E) isomer (here 22.55 min) following the USP monograph specification.

Table 3. Repeatability of doxepin HCl standard injections (0.1 mg/mL) - Peak areas and retention times

	(E)-Isomer		(Z)-Isomer
Injection	Peak Area (mAU*min)	RT  (min)	Peak Area (mAU*min)	RT (min)
1st	31.917	22.55	5.665	25.74
2nd	31.822	22.53	5.666	25.72
3rd	31.404	22.55	5.571	25.73
4th	31.319	22.58	5.563	25.75
5th	30.763	22.58	5.474	25.77
Mean	31.445	22.55	5.588	25.74
Standard Deviation	0.4603	0.02	0.0805	0.02
% RSD	1.47	0.10	1.44	0.08
USP Requirement (RSD)	NMT 2.0%		NMT 2.0%

Linearity & Sensitivity

The linearity of (E)- and (Z)-isomers was demonstrated for 8 concentrations from 0.01 to 1.0 mg/mL with an R² of 0.9999 (Tables 3 & 4, Figures 2 & 3). This is 10 to 1000% of the doxepin test concentration at 0.1 mg/mL. The compendial guideline for the establishment of an assay linearity is 5 concentrations from 80% to 120% of the test concentration. 

Table 4. Linearity and sensitivity (E)-isomer – Average peak areas of duplicates (0.01 to 1.0 mg/mL doxepin HCL RS injections)

Conc.* mg/mL	Mean Area (mAU*min)
0.0103	3.247
0.0256	8.237
0.0513	16.054
0.1026	31.613
0.2050	64.147
0.4102	127.352
0.8204	250.354
1.0255	315.945
STEYX	0.997
Slope	306.75
R2	0.9999
LOD* (mg/mL)	0.011
LOQ* (mg/mL)	0.033
*related to the used conc. of doxepin HCl RS

Figure 2. Calibration curve for (E)-isomer from 0.01 to 1.0 mg/mL doxepin HCL RS injections.

Table 5. Linearity and sensitivity for (Z)-isomer – Average peak area of duplicates (0.01 to 1.0 mg/mL doxepin HCL RS injections)

Conc.* mg/mL	Mean Area (mAU*min)
0.0103	0.577
0.0256	1.433
0.0513	2.832
0.1026	5.619
0.2050	11.418
0.4102	22.771
0.8204	44.825
1.0255	56.552
STEYX	0.167
Slope	54.951
R2	0.9999
LOD* (mg/mL)	0.010
LOQ* (mg/mL)	0.030
*Related to the used conc. of doxepin HCl RS

Figure 3. Calibration curve for (Z)-isomer from 0.01 to 1.0 mg/mL doxepin HCL RS injections.

Conclusion

The Purospher^® STAR RP-8e column was able to comply with the criteria set out for the assay of doxepin HCl in the USP monograph and demonstrated a solid performance:

• The resolution between (E)- and (Z)-isomers was 1.84. (criteria NLT 1.5)
• The peak symmetry factor of the doxepin (E)-isomer ranged from 0.96 to 1.32 for a concentration from 0.01 to 1 mg/mL. (criteria NMT 2.0)
• % RSD for 5 repeats at 0.1 mg/mL doxepin was 1.47 and 1.44 for the peak areas of the (E)- and (Z)-isomers respectively. (criteria NMT 2%)
• The retention time of (E)- and (Z)-isomers was 22.55 and 25.74 min respectively. (RSD < 2% for n=5)
• LOD and LOQ of the doxepin (E)-isomer was 0.011 and 0.033 mg/mL respectively.*
• LOD and LOQ of the doxepin (Z)-isomer was 0.010 and 0.030 mg/mL respectively.*

*related to the used conc. of doxepin HCl RS

The column was robust and worked well at the low pH of 2.5 at an elevated temperature of 50 °C. Extended repeated injections (data not shown) showed good reproducibility for the retention times so long as the pH is well controlled within ±0.1 units. The column provided excellent peak shapes and was linear for an extended test range – both of which is essential for accurate quantification. 

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References

1. PubChem. Doxepin. Nih.gov. . [Internet]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Doxepin

2. United States Pharmacopeia (2021). USP Monographs, Doxepin Hydrochloride. [Internet]. Rockville, MD: United States Pharmacopeia.[cited 11 Apr 2022]. Available from: https://doi.usp.org/USPNF/USPNF_M28110_07_01