Development and validation of GC-MS method for analysis of chloropyramine hydrochloride in ointments
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A gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the analysis of chloropyramine hydrochloride in ointments, demonstrating high sensitivity and reproducibility. The method's validation included assessments of linearity, precision, recovery, and limits of detection and quantification, with recoveries between 90% and 98.7%. This rapid and accurate method is suitable for routine analysis of chloropyramine hydrochloride in commercially marketed ointments.
![IOSR Journal Of Pharmacy
(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219
www.iosrphr.org Volume 5, Issue 5 (May 2015), PP. 55-59
55
Development and validation of GC-MS method for analysis of
chloropyramine hydrochloride in ointments
Vesna Kostik1*
, Biljana Gjorgeska2
, Sofija Petkovska2
1
Institute of Public Health of Republic of Macedonia, 50 Divizija No. 6 , 1000 Skopje, Republic of Macedonia
2
Faculty of Medicine, Department of Pharmacy, University Goce Delchev, Shtip, Republic of Macedonia
Abstract: A simple, rapid, sensitive, specific, accurate, and reproducible method for the determination of
chloropyramine hydrochloride in ointments based on gas chromatography – mass spectrometry detection was
developed and validated. Dissolution of chloropyramine hydrochloride in the ointment was performed with 0.5 M
HCl. After alkalization with 25% (V/V) NH4OH, chloropyramine base was extracted with chloroform. The method
was validated in respect of linearity, specificity, precision, recovery, limit of detection (LOD), limit of quantification
(LOQ) and stability. In terms of performances: Recovery 90.0 – 98.7%, LOD 0.04 mg/g, LOQ 0.132 mg/g, specificity,
selectivity and precision the proposed method was found suitable for routine analysis.
Keywords: chloropyramine hydrochloride, chloropyramine base, gas chromatography- mass detection, ointment
I. INTRODUCTION
Chloropyramine is a first generation antihistamine drug approved in some Eastern European countries
for the treatment of allergic conjunctivitis, allergic rhinitis, bronchial asthma, and other allergic conditions [1].
Chloropyramine is known as a competitive reversible H1-receptor antagonist. By blocking the effects of
histamine, the drug inhibits the vasodilatation, increased vascular permeability, and tissue edema associated
with histamine release in the tissue [2]. The use of topical antihistamines preparations represented a major
advance in dermatology. Chloropyramine hydrochloride (N-[(4-chlorophenyl)methyl]-N',N'-dimethyl-N-
pyridin-2-ylethane-1,2-diamine hydrochloride) is an antihistamine of ethylenediamine group (Figure 1)
indicated for the treatment of several pathologies due to its anti-allergic and anti-inflammatory effects [3, 4].
This antihistamine is frequently incorporated in ointments and creams [5].
Ointments are homogeneous, viscous, semi-solid preparation, most commonly greasy, thick oil (oil
80% - water 20%) with a high viscosity that is intended for external application to the skin or mucous
membranes. Ointments have a water number that defines the maximum amount of water that it can contain.
They are used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or
prophylactic purposes and where a degree of occlusion is desired [6]. The vehicle of an ointment is known as
the ointment base. Commonly they are molecules self-assembled in water or in oil, leading to the formation of a
well defined microstructure. These heterogeneous systems can interfere with drug separation and detection, and
an adequate analytical method is needed to analyze the drug carried by these systems.
Figure 1. Chemical structure of chloropyramine hydrochloride
Gravimetric and spectrophotometric methods have been described for the simultaneous determination
of chloropyramine, amitriptiline, imipramine and antazoline in coated tablets [7]. A novel High - performance
liquid chromatography method (HPLC) for the simultaneous determination of antihistamine bamipine,
sympathomimetic amines and dextromethorphan in bulk drug material have been developed [8]. HPLC method
was also reported for the determination of antihistamines pheniramine and pyrilamine in two over the counter
cold medications [9]. HPLC remains the analytical method of choice, especially for analysis of the topical
formulations, owing to their complex composition [10].](https://image.slidesharecdn.com/j055055059-150613092559-lva1-app6892/85/Development-and-validation-of-GC-MS-method-for-analysis-of-chloropyramine-hydrochloride-in-ointments-1-320.jpg)
![56
In the last two decades a remarkable advancement in the hyphenated techniques and its application in
pharmaceutical analysis have been achieved. A variety of hyphenated techniques such as liquid chromatography
tandem mass spectrometry (LC-MS), gas chromatography with mass detection (GC-MS) etc. have been applied
in the analysis of pharmaceuticals [11]. However, up to our knowledge, no GC-MS method for the analysis of
chloropyramine hydrochloride in ointments has been described. The aim of this study was to develop a simple,
rapid, specific, precise and accurate method based on GC-MS technique for the determination of
chloropyramine hydrochloride in ointments. The method was validated in respect of linearity, specificity,
precision, recovery, limit of detection (LOD), limit of quantification (LOQ) and stability [12, 13].
II. MATERIALS AND METHODS
2.1. Instrumentation and chromatographic conditions
The method was performed on a Shimadzu GC-MS model QP 2010 Ultra. Chromatographic separation
was achieved on a fused silica ZB-5 capillary column (30 m x 0.25 mm i.d. x 0.25 µm film thickness), supplied
by Phenomenex (Torrance, USA). Helium purity 99.99999% was used as carrier gas. Operating conditions are
given in Table 1.
Table 1. GC-MS operating conditions
Column oven T 180 0
C
Injection T 250 0
C
Injection mode Splitless
Sampling time 1 min
Flow control mode Pressure
Pressure 60 kPa
Column flow 0.62 mL/min
Linera velocity 30.2 cm/sec
Purge flow 6.0 ml/min
High pressure injection On
High pressure injection pressure 250 kPa
High pressure injection time 1.0 min
Oven temperature program
Rate T(0
C) Hold time (min)
- 180.0 0
5.00 240 15
Injector type PTV
PTV carrier Yes
PTV purge Yes
Equilibrium time 3.0 min
Ion source temperature 200 0
C
Interface temperature 250 0
C
Solvent cut time 1.0 min
Detector gain mode Relative
Detector gain 0.2 kV
MS table
Start time 11.92 min
End time 13.92 min
Acquisition mode Selected ion monitoring (SIM)
Event time 0.3 sec
Ch1-m/z 58
Ch2-m/z 125
Ch3-m/z 71.0
2.2. Reagents and standards
Chloropyramine hydrochloride purity ≥99.8% was obtained from Fluka, Germany Chloropyramine-
base purity ≥99.7% was obtained from Alkaloid, Macedonia. The ointment was composed of white wax (pellets
- Sigma Aldrich, Germany) and white petrolatum (Sigma Aldrich, Germany). Chloroform (Merck, Germany)
gas chromatography grade was used for the extraction and preparation of standard solutions. Ammonium
hydroxide (25%, V/V) was obtained from Fluka, Germany. Hydrochloric acid (37%, V/V) was obtained from
Sigma Aldrich, Germany. Anhydrous sodium sulfate was obtained from Merck, Germany. Water was obtained
by distillation.](https://image.slidesharecdn.com/j055055059-150613092559-lva1-app6892/85/Development-and-validation-of-GC-MS-method-for-analysis-of-chloropyramine-hydrochloride-in-ointments-2-320.jpg)
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2.3. Preparation of the ointment
All components, the white wax and the petrolatum, were accurately weighed. The white wax was
melted on a hot plate at 70 – 750
C. When the wax was completely melted, the petrolatum was added and the
entire mixture was put on a hot plate until liquefied. Following liquefaction, the mixture was removed from heat
and was allowed to congeal. The mixture was stirred until it begun to congeal. Drug-loaded ointments were
prepared by adding 1% (W/W) chloropyramine hydrochloride to the ointment at room temperature. The mixture
was gently shaken to ensure complete mixing and dissolution. The drug-containing ointments and solutions
were shielded from light by storing in flasks wrapped with aluminum foil.
2.4. Preparation of standard solutions
Chloropyramine standard stock solution with the concentration of 1mg/mL (i) was prepared in
chloroform by transferring 10 mg of chloropyramine standard accurately weighted to a 10 mL volumetric flask
and filled up with chloroform to the mark. An aliquot of 0.25 mL of the standard stock solution (i) was
transferred to a 25 mL volumetric flask and was diluted with chloroform to obtain the concentration of 40
μg/mL (standard stock solution ii).
Working standard solutions, in a concentration range of 0.4 – 4.0 μg/mL, were prepared by dilution of
the standard stock solution (ii) with chloroform. 1 μL of each concentration was injected into the GC-MS system
and the area under curve (AUC) for each peak was plotted versus chloropyramine concentration. The analyses
were carried out in triplicate and a straight line standard curve was obtained by linear regression of the
experimental data.
2.5. Sample preparation
An aliquot of 0.1 g of homogenized ointment which contains 1% (W/W) chloropyramine hydrochloride
was accurately weighed and dissolved in approximately 10 mL 0.5 M HCl. The solution was quantitatively
transferred into the 100 mL separatory funnel. pH value of the solution was adjusted to 9-10 with 25% NH4OH
(V/V). After alkalization chloropyramine-base was extracted with 40 mL chloroform. Chloroform layer was
transferred through anhydrous sodium sulfate into the 50 mL volumetric flask and was filled with chloroform up
to the mark. 1 mL of the solution was transferred into the 10 mL volumetric flask. The solution was filtered
through a 0.22 μm Millipore membrane filter. 2 mL of the filtrate were transferred into the auto - sampler vial. 1
μL were injected into the injector port of the GC-MS. All the determinations were conducted in triplicate.
2.6. Determination
Determination of chloropyramine was performed by GC-MS in SIM mode under the operating
instrumental conditions shown in Table 1. Quantification was made by comparison of area under the curve
(AUC) obtained for chloropyramine extract with AUC obtained for the corresponding analytical standards.
2.7. Validation of the method
The method was validated in accordance with International Conference on Harmonization guidelines
(ICH-2003) for validation of analytical procedures [12].
Specificity and Selectivity
These parameters were determined by comparing the chromatograms of the chloroporymanine
standards with the chromatograms obtained for drug-loaded ointment and ointment without drug.
Linearity
The linearity was determined from the analytical curves obtained by GC-MS analysis of
chloropyramine standard solutions.
Recovery experiment
Recovery was determined by the standard addition method. The ointment samples in which 2.5mg/g of
the chloropyramine hydrochloride had been incorporated previously were fortified with different amounts of
chloropyramine hydrochloride solution prepared in 0.5 M HCl. The final concentrations of the fortified samples
were 5 mg/g, 7.5 mg/g, 10 mg/g, 12.5 and 15 mg/g, respectively. The recovery experiments were performed in
triplicate for each concentration.
Precision
The precision of the assay was determined by repeatability (intra-day) and intermediate precision
(inter-day). The intra-day precision was calculated as relative standard deviation (RSD) of results from ten
standard samples, during the same day, and the inter-day precision was studied by comparing the assays on two
different days. Ten samples of ointment fortified with chloropyramine hydrochloride at 10 mg/g were prepared
and assayed, and the standard deviation (SD) and RSD were calculated.](https://image.slidesharecdn.com/j055055059-150613092559-lva1-app6892/85/Development-and-validation-of-GC-MS-method-for-analysis-of-chloropyramine-hydrochloride-in-ointments-3-320.jpg)
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Calculation of LOD and LOQ
LOD and the limit of quantification LOQ were calculated according to the formulas LOD = 3.3 ·
SD/slope and LOQ = 10 · SD/slope [14].
III. RESULTS AND DISCUSSION
3.1. Method optimization
Because of poor solubility of chloropyramine hydrochloride (salt) in water and in the majority of
organic solvents (polar and non-polar), in our experiment as a suitable solvent for chloropyramine hydrochloride
dissolution we used 0.5 M HCl. In their study, Iliaszenko et al. used 0.5 M HCl for dissolution of several
tertiary amine hydrochloride drugs in coated tablets, prior their spectrophotometric determination [7].
Chloropyramine base was deliberated from the salt by addition of 25% NH4OH (V/V) to pH 9-10.
Extraction of the free base was efficiently performed with chloroform.
In order to improve specificity and selectivity and minimize interferences from ointment or solvent
systems that may occur in spetrophotometric or HPLC determinations, we performed the analysis using GC-MS
system. Selectivity was obtained by operating in selected ion monitoring (SIM) mode with target ion (58 m/z)
and reference ions (71 m/z, 125 m/z). Figure 1 depicts the mass spectrum of chloropyramine hydrochloride.
Figure 2. Mass spectrum of chloropyramine hydrochloride
3.2. Method validation
Recovery, Precision and Linearity
Statistical data for mean recovery, precision data and linearity of the method for the determination of
chloropyramine hydrochloride in ointments are shown in Table 2.
Table 2. Statistical data for mean recovery, precision data and linearity of the method
Fortification level
(mg/g)
FA found - mean
value (mg/kg ± SD)
Recovery (%),
n=3
RSD (%) Regression equation
5.0 4.5 ± 0.12 90.0 2.67
y = 1.032x – 0.74
R2
= 0.9996
7.5 6.9 ± 0.18 92.0 2.61
10.0 9.6 ± 0.28 96.0 2.92
12.5 12.1 ±0.34 96.8 2.81
15.0 14.8 ± 0.32 98.7 2.16
From the obtained results, it can be noticed that the proposed method is accurate and precise enough for
the determination of chloropyramine hydrochloride in ointments. The obtained values for correlation coefficient
R2
= 0.9996 indicated that the method has a good linearity. High analytical recoveries ranging from 90% to
98.7% were obtained for chloropyramine hydrochloride determination.
Repeatability and Reproducibility
Statistical data for within day repeatability and between day reproducibility of the method for the
determination of chloropyramine hydrochloride are shown in Table 3.
Table 3. Statistical data for repeatability and reproducibility of the method
tR /min
Within day Repeatability
(RSD, %); n=10
Between day Reproducibility (RSD, %); n=25
12.941 0.112 3.27 0.190 4.28
50 100 150 200 250 300 350 400 450 500 550
0
25
50
75
100
%
58
125
71
219105 289254183162 341327 429415 451 470 505369 535](https://image.slidesharecdn.com/j055055059-150613092559-lva1-app6892/85/Development-and-validation-of-GC-MS-method-for-analysis-of-chloropyramine-hydrochloride-in-ointments-4-320.jpg)
![59
The calculated RSD values for retention time (tR) for the within day repeatability and between day
reproducibility were found to be 0.112% and 0.19%, respectively which indicated good precision of the tR. Good
precision was also obtained for the within day repeatability and between day reproducibility of the peak area
(RSD 3.27% - 4.28%).
Stability studies
Five aliquots of the extracts of quality control (QC) samples with the nominal concentration of 10mg/g
chloropyramine hydrochloride were stored at -200
C over the period of 12 weeks. It was found that extracts were
stable within a period of 4 weeks. Figure 3 shows an overlay of chromatograms of extracts of chloropyramine
base in chloroform over the period of 4, 8 and 12 weeks, respectively.
Figure 3. Overlay of chromatograms of extracts of chloropyramine base in chloroform (1-after 4
weeks, 2- after 8 weeks and 3-after 12 weeks of storage at -200
C).
With the proposed methods the calculated values for LOD and LOQ were 0.04 mg/g and 0.132 mg/g,
respectively.
IV. CONCLUSION
In summary, the described method is rapid, sensitive, specific, accurate, and reproducible. It was
successfully used for routine determinations of chloropyramine hydrochloride in commercially marketed
ointments.
REFERENCES
[1]. J. R Vaughan, G. W. Anderson, R. C. Clapp, J. H Clark, J. P. English, K. L Howard,. H. W. Marson, L. H. Sutherland and J. J.
Denton, Antihistamine agents; halogenated N,N-dimethyl-N-benzyl-N-(2-pyridyl)-ethylenediamines, Journal of Organic
Chemistry 14 (2), 1949, 228–234. doi:10.1021/jo01154a006.
[2]. B.S. Deshmanker, S. L Agarwal, The action of three recent antihistaminic drugs--ambodryl, sandosten and synopen on
bronchial muscle, Indian J Med Sci. 13, 1959, 762-766. PMID: 13816133
[3]. Kurenova EV, Hunt DL, He D, Magis AT, Ostrov DA, Cance WG: Small molecule chloropyramine hydrochloride (C4) targets
the binding site of focal adhesion kinase and vascular endothelial growth factor receptor 3 and suppresses breast cancer growth in
vivo. J Med Chem. 2009 Aug 13;52(15):4716-24. doi: 10.1021/jm900159g.
[4]. G. Wilhelmi, The analgesic effects of anti-inflammatory drugs from the point of view of different pharmacological test methods.
Acta Med. Okayama, 18, 1964, 297-310.
[5]. D. Zaghi, H.I. Maibach, Survey of Safety and efficacy information in drug inserts for topical prescription
medications". American Journal of Clinical Dermatology 8(1), 2007, 43–46. doi:10.2165/00128071-200708010-00006.
[6]. S. E Wolverton, Comprehensive Dermatologic Drug Therapy. WB Saunders. 2001, 563-572.
[7]. J. Iliaszenko, M. Sokolowska, I. Szlaska and R. Paruszewski, Gravimetric and spectrophotometric determination of some drugs-
tertiary amine hydrochlorides in coated tablets, Acta Polonia Pharmaceutica – Dug research, 57(2), 2000, 93-95.
[8]. J. E. Kountourellis, C. K. Markopoulou and P. P. Georgakopoulos, An HPLC Method for the Separation and Simultaneous
Determination of Antihistamines, Sympathomimetic Amines and Dextromethorphan in Bulk Drug Material and Dosage Forms,
Analytical Letters, 23(5), 1990, 883-891. doi: 10.1080/00032719008052489.
[9]. D. Rudolph and L. Holkup, HPLC determination and quantification of pheniramine and pyrilamine in over the counter cold
medicines, Concordia College Journal of Analytical Chemistry, 1, 2010, 29-33.
[10]. M.C.C. Urban, R.M. Mainardes, and M.P.Daflon, Development and validation of HPLC method for analysis of dexamethsone
acetate in microemulsions, Brazilian Journal of Pharmaceutical Science, 45(1), 2009, 87-92.
[11]. M. R. Siddiqui., Z.A AlOthman and N. Rahman, Analytical techniques in pharmaceutical analysis: A review, Arabian Journal of
chemistry, 2013, http://dx.doi.org/10.1016/j.arabjc.2013.04.016
[12]. INTERNATIONAL CONFERENCE ON HARMONIATION (ICH); validation of analytical procedures: Methodology, Q2B
(CPMP/ICH/281/95), 2003. Available at: http://www.ich.org. Last access on: 8th. Feb. 2015.
[13]. BRITISH PHARMACOPOEIA. London: The Stationery Office, 2, 2001, p.A437-A8.
[14] J.C. Miller. and J. N. Miller, Statistic for analytical chemistry, 3rd Ed., Ellis Horwood Ptr. Prentice Hall, 1993, 104 – 141.](https://image.slidesharecdn.com/j055055059-150613092559-lva1-app6892/85/Development-and-validation-of-GC-MS-method-for-analysis-of-chloropyramine-hydrochloride-in-ointments-5-320.jpg)
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Development and validation of GC-MS method for analysis of chloropyramine hydrochloride in ointments
- 1. IOSR Journal Of Pharmacy (e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219 www.iosrphr.org Volume 5, Issue 5 (May 2015), PP. 55-59 55 Development and validation of GC-MS method for analysis of chloropyramine hydrochloride in ointments Vesna Kostik1* , Biljana Gjorgeska2 , Sofija Petkovska2 1 Institute of Public Health of Republic of Macedonia, 50 Divizija No. 6 , 1000 Skopje, Republic of Macedonia 2 Faculty of Medicine, Department of Pharmacy, University Goce Delchev, Shtip, Republic of Macedonia Abstract: A simple, rapid, sensitive, specific, accurate, and reproducible method for the determination of chloropyramine hydrochloride in ointments based on gas chromatography – mass spectrometry detection was developed and validated. Dissolution of chloropyramine hydrochloride in the ointment was performed with 0.5 M HCl. After alkalization with 25% (V/V) NH4OH, chloropyramine base was extracted with chloroform. The method was validated in respect of linearity, specificity, precision, recovery, limit of detection (LOD), limit of quantification (LOQ) and stability. In terms of performances: Recovery 90.0 – 98.7%, LOD 0.04 mg/g, LOQ 0.132 mg/g, specificity, selectivity and precision the proposed method was found suitable for routine analysis. Keywords: chloropyramine hydrochloride, chloropyramine base, gas chromatography- mass detection, ointment I. INTRODUCTION Chloropyramine is a first generation antihistamine drug approved in some Eastern European countries for the treatment of allergic conjunctivitis, allergic rhinitis, bronchial asthma, and other allergic conditions [1]. Chloropyramine is known as a competitive reversible H1-receptor antagonist. By blocking the effects of histamine, the drug inhibits the vasodilatation, increased vascular permeability, and tissue edema associated with histamine release in the tissue [2]. The use of topical antihistamines preparations represented a major advance in dermatology. Chloropyramine hydrochloride (N-[(4-chlorophenyl)methyl]-N',N'-dimethyl-N- pyridin-2-ylethane-1,2-diamine hydrochloride) is an antihistamine of ethylenediamine group (Figure 1) indicated for the treatment of several pathologies due to its anti-allergic and anti-inflammatory effects [3, 4]. This antihistamine is frequently incorporated in ointments and creams [5]. Ointments are homogeneous, viscous, semi-solid preparation, most commonly greasy, thick oil (oil 80% - water 20%) with a high viscosity that is intended for external application to the skin or mucous membranes. Ointments have a water number that defines the maximum amount of water that it can contain. They are used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or prophylactic purposes and where a degree of occlusion is desired [6]. The vehicle of an ointment is known as the ointment base. Commonly they are molecules self-assembled in water or in oil, leading to the formation of a well defined microstructure. These heterogeneous systems can interfere with drug separation and detection, and an adequate analytical method is needed to analyze the drug carried by these systems. Figure 1. Chemical structure of chloropyramine hydrochloride Gravimetric and spectrophotometric methods have been described for the simultaneous determination of chloropyramine, amitriptiline, imipramine and antazoline in coated tablets [7]. A novel High - performance liquid chromatography method (HPLC) for the simultaneous determination of antihistamine bamipine, sympathomimetic amines and dextromethorphan in bulk drug material have been developed [8]. HPLC method was also reported for the determination of antihistamines pheniramine and pyrilamine in two over the counter cold medications [9]. HPLC remains the analytical method of choice, especially for analysis of the topical formulations, owing to their complex composition [10].
- 2. 56 In the last two decades a remarkable advancement in the hyphenated techniques and its application in pharmaceutical analysis have been achieved. A variety of hyphenated techniques such as liquid chromatography tandem mass spectrometry (LC-MS), gas chromatography with mass detection (GC-MS) etc. have been applied in the analysis of pharmaceuticals [11]. However, up to our knowledge, no GC-MS method for the analysis of chloropyramine hydrochloride in ointments has been described. The aim of this study was to develop a simple, rapid, specific, precise and accurate method based on GC-MS technique for the determination of chloropyramine hydrochloride in ointments. The method was validated in respect of linearity, specificity, precision, recovery, limit of detection (LOD), limit of quantification (LOQ) and stability [12, 13]. II. MATERIALS AND METHODS 2.1. Instrumentation and chromatographic conditions The method was performed on a Shimadzu GC-MS model QP 2010 Ultra. Chromatographic separation was achieved on a fused silica ZB-5 capillary column (30 m x 0.25 mm i.d. x 0.25 µm film thickness), supplied by Phenomenex (Torrance, USA). Helium purity 99.99999% was used as carrier gas. Operating conditions are given in Table 1. Table 1. GC-MS operating conditions Column oven T 180 0 C Injection T 250 0 C Injection mode Splitless Sampling time 1 min Flow control mode Pressure Pressure 60 kPa Column flow 0.62 mL/min Linera velocity 30.2 cm/sec Purge flow 6.0 ml/min High pressure injection On High pressure injection pressure 250 kPa High pressure injection time 1.0 min Oven temperature program Rate T(0 C) Hold time (min) - 180.0 0 5.00 240 15 Injector type PTV PTV carrier Yes PTV purge Yes Equilibrium time 3.0 min Ion source temperature 200 0 C Interface temperature 250 0 C Solvent cut time 1.0 min Detector gain mode Relative Detector gain 0.2 kV MS table Start time 11.92 min End time 13.92 min Acquisition mode Selected ion monitoring (SIM) Event time 0.3 sec Ch1-m/z 58 Ch2-m/z 125 Ch3-m/z 71.0 2.2. Reagents and standards Chloropyramine hydrochloride purity ≥99.8% was obtained from Fluka, Germany Chloropyramine- base purity ≥99.7% was obtained from Alkaloid, Macedonia. The ointment was composed of white wax (pellets - Sigma Aldrich, Germany) and white petrolatum (Sigma Aldrich, Germany). Chloroform (Merck, Germany) gas chromatography grade was used for the extraction and preparation of standard solutions. Ammonium hydroxide (25%, V/V) was obtained from Fluka, Germany. Hydrochloric acid (37%, V/V) was obtained from Sigma Aldrich, Germany. Anhydrous sodium sulfate was obtained from Merck, Germany. Water was obtained by distillation.
- 3. 57 2.3. Preparation of the ointment All components, the white wax and the petrolatum, were accurately weighed. The white wax was melted on a hot plate at 70 – 750 C. When the wax was completely melted, the petrolatum was added and the entire mixture was put on a hot plate until liquefied. Following liquefaction, the mixture was removed from heat and was allowed to congeal. The mixture was stirred until it begun to congeal. Drug-loaded ointments were prepared by adding 1% (W/W) chloropyramine hydrochloride to the ointment at room temperature. The mixture was gently shaken to ensure complete mixing and dissolution. The drug-containing ointments and solutions were shielded from light by storing in flasks wrapped with aluminum foil. 2.4. Preparation of standard solutions Chloropyramine standard stock solution with the concentration of 1mg/mL (i) was prepared in chloroform by transferring 10 mg of chloropyramine standard accurately weighted to a 10 mL volumetric flask and filled up with chloroform to the mark. An aliquot of 0.25 mL of the standard stock solution (i) was transferred to a 25 mL volumetric flask and was diluted with chloroform to obtain the concentration of 40 μg/mL (standard stock solution ii). Working standard solutions, in a concentration range of 0.4 – 4.0 μg/mL, were prepared by dilution of the standard stock solution (ii) with chloroform. 1 μL of each concentration was injected into the GC-MS system and the area under curve (AUC) for each peak was plotted versus chloropyramine concentration. The analyses were carried out in triplicate and a straight line standard curve was obtained by linear regression of the experimental data. 2.5. Sample preparation An aliquot of 0.1 g of homogenized ointment which contains 1% (W/W) chloropyramine hydrochloride was accurately weighed and dissolved in approximately 10 mL 0.5 M HCl. The solution was quantitatively transferred into the 100 mL separatory funnel. pH value of the solution was adjusted to 9-10 with 25% NH4OH (V/V). After alkalization chloropyramine-base was extracted with 40 mL chloroform. Chloroform layer was transferred through anhydrous sodium sulfate into the 50 mL volumetric flask and was filled with chloroform up to the mark. 1 mL of the solution was transferred into the 10 mL volumetric flask. The solution was filtered through a 0.22 μm Millipore membrane filter. 2 mL of the filtrate were transferred into the auto - sampler vial. 1 μL were injected into the injector port of the GC-MS. All the determinations were conducted in triplicate. 2.6. Determination Determination of chloropyramine was performed by GC-MS in SIM mode under the operating instrumental conditions shown in Table 1. Quantification was made by comparison of area under the curve (AUC) obtained for chloropyramine extract with AUC obtained for the corresponding analytical standards. 2.7. Validation of the method The method was validated in accordance with International Conference on Harmonization guidelines (ICH-2003) for validation of analytical procedures [12]. Specificity and Selectivity These parameters were determined by comparing the chromatograms of the chloroporymanine standards with the chromatograms obtained for drug-loaded ointment and ointment without drug. Linearity The linearity was determined from the analytical curves obtained by GC-MS analysis of chloropyramine standard solutions. Recovery experiment Recovery was determined by the standard addition method. The ointment samples in which 2.5mg/g of the chloropyramine hydrochloride had been incorporated previously were fortified with different amounts of chloropyramine hydrochloride solution prepared in 0.5 M HCl. The final concentrations of the fortified samples were 5 mg/g, 7.5 mg/g, 10 mg/g, 12.5 and 15 mg/g, respectively. The recovery experiments were performed in triplicate for each concentration. Precision The precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day). The intra-day precision was calculated as relative standard deviation (RSD) of results from ten standard samples, during the same day, and the inter-day precision was studied by comparing the assays on two different days. Ten samples of ointment fortified with chloropyramine hydrochloride at 10 mg/g were prepared and assayed, and the standard deviation (SD) and RSD were calculated.
- 4. 58 Calculation of LOD and LOQ LOD and the limit of quantification LOQ were calculated according to the formulas LOD = 3.3 · SD/slope and LOQ = 10 · SD/slope [14]. III. RESULTS AND DISCUSSION 3.1. Method optimization Because of poor solubility of chloropyramine hydrochloride (salt) in water and in the majority of organic solvents (polar and non-polar), in our experiment as a suitable solvent for chloropyramine hydrochloride dissolution we used 0.5 M HCl. In their study, Iliaszenko et al. used 0.5 M HCl for dissolution of several tertiary amine hydrochloride drugs in coated tablets, prior their spectrophotometric determination [7]. Chloropyramine base was deliberated from the salt by addition of 25% NH4OH (V/V) to pH 9-10. Extraction of the free base was efficiently performed with chloroform. In order to improve specificity and selectivity and minimize interferences from ointment or solvent systems that may occur in spetrophotometric or HPLC determinations, we performed the analysis using GC-MS system. Selectivity was obtained by operating in selected ion monitoring (SIM) mode with target ion (58 m/z) and reference ions (71 m/z, 125 m/z). Figure 1 depicts the mass spectrum of chloropyramine hydrochloride. Figure 2. Mass spectrum of chloropyramine hydrochloride 3.2. Method validation Recovery, Precision and Linearity Statistical data for mean recovery, precision data and linearity of the method for the determination of chloropyramine hydrochloride in ointments are shown in Table 2. Table 2. Statistical data for mean recovery, precision data and linearity of the method Fortification level (mg/g) FA found - mean value (mg/kg ± SD) Recovery (%), n=3 RSD (%) Regression equation 5.0 4.5 ± 0.12 90.0 2.67 y = 1.032x – 0.74 R2 = 0.9996 7.5 6.9 ± 0.18 92.0 2.61 10.0 9.6 ± 0.28 96.0 2.92 12.5 12.1 ±0.34 96.8 2.81 15.0 14.8 ± 0.32 98.7 2.16 From the obtained results, it can be noticed that the proposed method is accurate and precise enough for the determination of chloropyramine hydrochloride in ointments. The obtained values for correlation coefficient R2 = 0.9996 indicated that the method has a good linearity. High analytical recoveries ranging from 90% to 98.7% were obtained for chloropyramine hydrochloride determination. Repeatability and Reproducibility Statistical data for within day repeatability and between day reproducibility of the method for the determination of chloropyramine hydrochloride are shown in Table 3. Table 3. Statistical data for repeatability and reproducibility of the method tR /min Within day Repeatability (RSD, %); n=10 Between day Reproducibility (RSD, %); n=25 12.941 0.112 3.27 0.190 4.28 50 100 150 200 250 300 350 400 450 500 550 0 25 50 75 100 % 58 125 71 219105 289254183162 341327 429415 451 470 505369 535
- 5. 59 The calculated RSD values for retention time (tR) for the within day repeatability and between day reproducibility were found to be 0.112% and 0.19%, respectively which indicated good precision of the tR. Good precision was also obtained for the within day repeatability and between day reproducibility of the peak area (RSD 3.27% - 4.28%). Stability studies Five aliquots of the extracts of quality control (QC) samples with the nominal concentration of 10mg/g chloropyramine hydrochloride were stored at -200 C over the period of 12 weeks. It was found that extracts were stable within a period of 4 weeks. Figure 3 shows an overlay of chromatograms of extracts of chloropyramine base in chloroform over the period of 4, 8 and 12 weeks, respectively. Figure 3. Overlay of chromatograms of extracts of chloropyramine base in chloroform (1-after 4 weeks, 2- after 8 weeks and 3-after 12 weeks of storage at -200 C). With the proposed methods the calculated values for LOD and LOQ were 0.04 mg/g and 0.132 mg/g, respectively. IV. CONCLUSION In summary, the described method is rapid, sensitive, specific, accurate, and reproducible. It was successfully used for routine determinations of chloropyramine hydrochloride in commercially marketed ointments. REFERENCES [1]. J. R Vaughan, G. W. Anderson, R. C. Clapp, J. H Clark, J. P. English, K. L Howard,. H. W. Marson, L. H. Sutherland and J. J. Denton, Antihistamine agents; halogenated N,N-dimethyl-N-benzyl-N-(2-pyridyl)-ethylenediamines, Journal of Organic Chemistry 14 (2), 1949, 228–234. doi:10.1021/jo01154a006. [2]. B.S. Deshmanker, S. L Agarwal, The action of three recent antihistaminic drugs--ambodryl, sandosten and synopen on bronchial muscle, Indian J Med Sci. 13, 1959, 762-766. PMID: 13816133 [3]. Kurenova EV, Hunt DL, He D, Magis AT, Ostrov DA, Cance WG: Small molecule chloropyramine hydrochloride (C4) targets the binding site of focal adhesion kinase and vascular endothelial growth factor receptor 3 and suppresses breast cancer growth in vivo. J Med Chem. 2009 Aug 13;52(15):4716-24. doi: 10.1021/jm900159g. [4]. G. Wilhelmi, The analgesic effects of anti-inflammatory drugs from the point of view of different pharmacological test methods. Acta Med. Okayama, 18, 1964, 297-310. [5]. D. Zaghi, H.I. Maibach, Survey of Safety and efficacy information in drug inserts for topical prescription medications". American Journal of Clinical Dermatology 8(1), 2007, 43–46. doi:10.2165/00128071-200708010-00006. [6]. S. E Wolverton, Comprehensive Dermatologic Drug Therapy. WB Saunders. 2001, 563-572. [7]. J. Iliaszenko, M. Sokolowska, I. Szlaska and R. Paruszewski, Gravimetric and spectrophotometric determination of some drugs- tertiary amine hydrochlorides in coated tablets, Acta Polonia Pharmaceutica – Dug research, 57(2), 2000, 93-95. [8]. J. E. Kountourellis, C. K. Markopoulou and P. P. Georgakopoulos, An HPLC Method for the Separation and Simultaneous Determination of Antihistamines, Sympathomimetic Amines and Dextromethorphan in Bulk Drug Material and Dosage Forms, Analytical Letters, 23(5), 1990, 883-891. doi: 10.1080/00032719008052489. [9]. D. Rudolph and L. Holkup, HPLC determination and quantification of pheniramine and pyrilamine in over the counter cold medicines, Concordia College Journal of Analytical Chemistry, 1, 2010, 29-33. [10]. M.C.C. Urban, R.M. Mainardes, and M.P.Daflon, Development and validation of HPLC method for analysis of dexamethsone acetate in microemulsions, Brazilian Journal of Pharmaceutical Science, 45(1), 2009, 87-92. [11]. M. R. Siddiqui., Z.A AlOthman and N. Rahman, Analytical techniques in pharmaceutical analysis: A review, Arabian Journal of chemistry, 2013, http://dx.doi.org/10.1016/j.arabjc.2013.04.016 [12]. INTERNATIONAL CONFERENCE ON HARMONIATION (ICH); validation of analytical procedures: Methodology, Q2B (CPMP/ICH/281/95), 2003. Available at: http://www.ich.org. Last access on: 8th. Feb. 2015. [13]. BRITISH PHARMACOPOEIA. London: The Stationery Office, 2, 2001, p.A437-A8. [14] J.C. Miller. and J. N. Miller, Statistic for analytical chemistry, 3rd Ed., Ellis Horwood Ptr. Prentice Hall, 1993, 104 – 141.