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A rapid ultra-performance liquid chromatography (UPLC) method for the Women who give birth preterm commonly suffer with inade-
determination of domperidone in the presence of its process impurities quate production of breast milk. Asztalos et al. (4) is currently
and droperidol was developed and validated. The rapid chromatograph- investigating the use of domperidone in the enhancement of
ic separation was achieved using a sub 2 mm Hypersil Zorbax eXtra breast milk in women who have given birth to preterm babies.
Densely Bonded C18 column (30 3 4.6 mm, i.d., 1.8 mm). A gradient Women who have given birth by cesarean section also often
mobile phase consisting of Solvent A: 0.06 M ammonium acetate and fail to produce a sufficient amount of breast milk.
Solvent B: methanol, with a flow rate of 1 mL/min was employed. The Jantarasaengaram et al. (5) found that treating these women
column temperature was set at 4088 C, and the diode-array detector with domperidone tablets increased their milk production con-
was set at 280 nm. An injection volume of 3 mL was used. The current- siderably. Knoppert et al. (6) studied the optimal dosage of dom-
ly utilized European Pharmacopeia (Eur. Pharm.) method employed by peridone given to this particular demographic and discovered
Janssen Pharmaceuticals Ltd was run on a Hypersil Base-Deactivated over a 4-week period, a 20 mg dose of domperidone, three
Silica C18 column (100 3 4.6 mm, i.d., 3 mm) with a run time of times daily steadily increased their milk flow. Zavitsanos et al.
12.5 min. The developed UPLC method, with a run time of 7.5 min (7) developed a sensitive HPLC – mass spectrometry method to
was determined to be accurate, precise, specific, robust and highly determine domperidone in human blood plasma and in human
sensitive according to the International Conference on Harmonization breast milk. No previous study had been undertaken to deter-
guidelines. The method herein demonstrated a reduction in analysis mine domperidone in breast milk, and it was discovered that
time of 40%, allowing for a much higher sample throughput. A solvent domperidone does cross the blood barrier into breast milk but
consumption decrease of over 58% was also observed, which results in to a lesser extent than other galactagogue agents and therefore
a dramatic reduction in running costs for Janssen Pharmaceuticals Ltd. reduces the risk of toxicity to the mother and baby (8).
Domperidone has been examined by several authors within
the literature, in numerous different matrices and using many dif-
ferent liquid chromatography (LC) methods; Michaud et al. (9)
Introduction developed a sensitive LC method to determine domperidone
Domperidone, 5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1H-benzimi- and its major metabolites formed in vitro by human liver micro-
dazol-1-yl)propyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol- somes. Ali et al. (10) employed HPLC to concurrently analyze
2-one, is an antidopaminergic agent. It acts by preventing dopa- methyl- and propyl-paraben and domperidone in oral suspension.
mine binding to the receptors at the chemoreceptor trigger Ali et al. (11) employed HPLC to detect domperidone in the
zone. Domperidone is commonly employed as an antiemetic wastewater runoff of certain pharmaceutical companies.
drug used to prevent nausea and vomiting (1) and is the active Several authors including Wang et al. and Xu et al. have devel-
ingredient in motilium tablets and suppositories. It also acts as oped ultra-performance liquid chromatography – tandem mass
a prokinetic agent and enhances the strength of the esophageal spectrometric (UPLC – MS-MS) methods to determine domperi-
sphincter and is used in the treatment of gastroparesis (delayed done in blood plasma (12, 13). However, very little work has
gastric emptying) (2, 3). Lately, domperidone has taken preva- been done on the development and validation of a method for
lence as a galactagogue ( promotes lactation) and has been the examination of impurities in the bulk product. One such
used to increase breast milk production in women who have a paper similar to the present study by Seshadri et al. (14) devel-
low milk count (4, 5). Droperidol, 2H-benzimidazol-2-one,1-[1- oped a method for the estimation of impurities in the pharma-
[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6-tetrahydro-4-pyridinyl]-1, ceutical dosage form of omeprazole and domperidone
3-dihydro-1-1-[3-( p-fluorobenzoyl)propyl]-1,2,[3,6-tetrahydro-4- capsules. The newly developed method offers a much reduced
pyridyl]-2-benzimidazolinone, is also an antidopaminergic agent. analysis time; between 2 and 7 times faster, when compared
It is utilized within this study as a resolution check for with these published works. It also has the advantage of employ-
domperidone. ing a sub 2 mm column as opposed to traditional columns used in
The HPLC method employed by Janssen Pharmaceuticals to these articles.
determine domperidone in its active pharmaceutical ingredient The primary target of this work was to develop and validate a
(API) form and in the presence of its six impurities and droper- rapid method to determine domperidone in the presence of its pro-
idol is found in the Eur. Pharm (Figure 1). As well as being a cess impurities and make it suitable for immediate use by Janssen
worldwide prokinetic and antiemetic drug, domperidone is cur- Pharmaceuticals Ltd. The new rapid method cuts time, which in-
rently being investigated in extensive studies as a galactagogue. creases sample throughput and decreases solvent consumption.
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(A) Gradient elution for original method (B) Gradient elution for optimized
method
Time %B (MEOH) Time %B (MEOH)
0 30 0 30
10 100 5.5 100
12 100 6.5 100
12.5 30 7.0 30
7.5 30
Chemical names
All standards are CRM.
(i) Domperidone: 5-chloro-1-(1-[3-(2-oxo-2,3-dihydro-1
H-benzo[d]imidazol-1-yl)propyl]piperidin-4-yl)-1H-benzo[d]imi-
dazol-2(3H)-one; (ii) droperidol: 1-f1-[4-(4-fluorophenyl)-4-
oxobutyl]-1,2,5,6-tetrahydropyridin-4-yl]-1,3-dihydro-2H-benzi-
midazol-2-one; (iii) impurity A: 5-chloro-1-( piperidin-4-yl)-1,
3-dihydro-2H-benzimidazol) 2-one; (iv) impurity B: 4-(5-chloro-
2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)-1-formylpiperidin; (v)
impurity C: cis-4-(5-chloro-2-oxo-2,3-dihydro-1H-benzimidazol-
1-yl)-1-[3-(2-oxo-2,3-dihydro-1H-benzimidazol-1yl)propyl]pi-
peridine 1-oxide; (vi) impurity D: 5-chloro-3-[3-(2-oxo-2,
3-dihydro-1H-benzimidazol-1-yl)propyl]-1-[1-[3-(2-oxo-2,3-dihydro-
1H-benzimidazol-1-yl)propyl]piperidin-4-yl]-1,3-dihydro-2H-
benzimidazol-2-one; (vii) impurity E: 1-[3-[4-(5-chloro-2-oxo-2,
3-dihydro-1H-benzimidazol-1-yl)piperidin-1-yl]propyl]-3-[3-(2-
oxo-2,3-dihydro-1H-benzimidazol-1-yl)propyl]-1,3-dihydro-2H-
benzimidazol-2-one and (viii) impurity F: 1,3-bis[3-[4-(5-chloro-
2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-yl]propyl]-
1,3-dihydro-2H-benzimidazol-2-one.
Instrumentation
An Agilent 1200 Rapid Resolution Liquid Chromatography sys-
tem was used for this analysis. It contains a 1200 series binary
pump SL, a vacuum degasser, a 1200 series high-performance
auto sampler, a 1200 series thermostatted column compartment
SL and a 1200 series diode-array detector (DAD) SL for up to
80 Hz operation that were controlled by ChemStation B.02.01
SR1 data acquisition and evaluation software.
Figure 1. Chemical structure of domperidone, its six impurities and droperidol.
The laboratory stability studies indicate that domperidone and its Chromatographic conditions
impurities remain stable for up to 48 h; therefore, allowing for lon- Table I(A) illustrates the gradient elution program for the elution
ger sequences to be run than previously undertaken. of domperidone and its impurities. A 3 mm Hypersil Base-
Deactivated Silica (BDS) C18 column (100 4.6 mm, i.d., 3 mm)
was employed in the current European Pharmacopeia method
Experimental employed by Janssen. Table I(B) illustrates the developed gradi-
Chemicals and reagents ent elution program for the elution of domperidone and its
Samples of domperidone standard (API), its six impurities and dro- impurities utilizing the sub 2 mm Hypersil eXtra Densely
peridol standard were received from Janssen Pharmaceutical Ltd, Bonded (XDB) C18 column (30 4.6 mm, i.d., 1.8 mm). An
try and separate these two impurities but to not avail. The orig- Table II
inal flow rate of 1.5 mL/min was reduced to 1 mL/min. This was Linearity Data for Domperidone, Its Process-Related Impurities and Droperidol
to improve resolution, peak shape and also to reduce pressure. Compound Slope Y-intercept Correlation coefficient
The gradient steps were adjusted in increments to try and
Imp A 2.43 0.37 0.9998
achieve optimum separation in the shortest time possible. Imp B 2.88 0.71 0.9999
Increasing the temperature to 408C that lowered the back pres- Imp C 2.92 1.10 0.9999
Imp D&E 6.37 3.43 0.9995
sure and reduced the retention time of the API. The optimal gra- Imp F 2.74 1.04 0.9998
dient conditions for the new rapid method were as per Domperidone 3.80 0.98 0.9999
Table I(B), with a flow rate of 1 mL/min. The DAD was set to Droperidol 2.80 0.45 0.9999
280 nm, and the column temperature was set to 408C. An injec-
tion volume of 3 mL was used. The retention times of domperi-
done, its six impurities and droperidol were as follows:
impurity A: 1.29 min, impurity B: 2.57 min, domperidone: Table III
Recovery Data for Domperidone
3.93 min, droperidol: 4.11 min, impurity C: 5.01 min, impurities
D&E: 4.43 min and impurity F: 4.85 min (Figure 2). Concentration (mg/mL) %Recovery
15 101
25 101
35 98.9
Method Validation 45 99.6
Linearity 55 98.0
Table II
Table IV
Accuracy LOD and LOQ Values for Domperidone, Its Related Impurities and Droperidol
Figure 3. The resulting chromatogram for selectivity under optimum conditions using the sub 2 mm XDB column [15 mg/mL, chromatographic conditions as in Table I(B)].
Table VI
ambient temperature. According to the pharmacopeia mono-
% Difference for Laboratory Stability Studies after 48 h graph domperidone elutes at 6.5 min.
Following some initial experimental work and an extensive
Compound Clear vial Brown vial Clear vial ambient Brown vial ambient
48C 48C temperature temperature
survey of available columns, an XDB sub 2 mm column was cho-
sen. This column had similar specification, i.e., % carbon and sur-
Imp A 0.09 0.04 1.15 1.50
Imp B 0.33 0.31 1.83 1.92
face area to the BDS column. As discussed in Results section, the
Imp C 0.18 0.17 1.75 1.99 method was optimized by altering mobile phase, flow rate, col-
Imp D&E 0.16 0.15 1.50 1.68 umn temperature and gradient steepness on separate occasions
Imp F 0.28 0.20 1.63 1.70
Domperidone 0.21 0.19 1.48 1.56 and in small increments. After rigorous testing, with the aim of
Droperidol 0.12 0.07 1.35 1.49 achieving the fastest conceivable time and best possible resolu-
tion a gradient as per Table I(B) was established. The 7.5 min
run time included a 1 min wash stop and 0.5 min reequilibration
Sample stability
step. A flow rate of 1 mL/min, injection volume of 3 mL, column
Table VI
temperature of 408C and wavelength set at 280 nm were em-
ployed. Resolution between the critical pair’s domperidone
and droperidol and impurities F and C were 2.32 and 2.16, re-
Discussion spectively, .2.0 set out by Eur. Pharm. (Figure 2).
The purpose of this study was to develop and optimize a rapid The new rapid UPLC method demonstrates far superior anal-
method with significant reduction in analysis time while main- ysis times to the pharmacopeia method and also the articles re-
taining efficiency and to validate this rapid UPLC method accord- ported within this study, see Table VII. As mentioned in
ing to the ICH guidelines. Introduction section, several authors have developed LC meth-
The original domperidone method utilized by Janssen em- ods for domperidone analysis in many different matrices;
ployed a Hypersil BDS C18 column (100 4.6 mm, i.d., 3 mm) however, limited literature is available on the separation of dom-
with a run time of 12.5 min and a flow rate of 1.5 mL/min. The peridone and its impurities in the bulk formation. One compara-
original injection volume was 10 mL, and the column was at ble study by Seshadri et al. (14) developed and validated a
Authors Curtin et al. Eur. Phar. Michaud et al. Ali et al. Ali et al. Seshadri et al.
Analysis time (min) 7.5 12.5 13 12 20 55
Domperidone retention time (min) 3.9 6.5 12.2 8.4 8.29 31.6
Column Sub 2 mm XDB C18 BDS C18 3 mm ODS, C18 5 mm 5 mm, OP, C8 Waters symmetry 5 mm, C18 ODS 5 mm C18
Injection volume (mL) 2 10 10 –30 10 5 Not given
Method Validation
Sample stability
The new UPLC method was validated under the following param- As can be seen from Table VI, all compounds remained stable for
eters according to the ICH guidelines: linearity, accuracy, preci- 48 h in all conditions proving that domperidone and its impuri-
sion, LOD and LOQ, selectivity, robustness and some stability ties can now be analyzed in long sequences without sample deg-
studies were undertaken. radation taking place. Marginal differences in degradation were
detected between clear and amber vials at both temperatures,
and significant differences, however, were detected between
Linearity the 48C and ambient temperatures. After 48 h under ambient
Calibration curves for domperidone, its impurities and droperi- temperatures, degradation was only just below Janssen’s recom-
dol all gave a linear response over the range of 15 – 55 mg/mL. mended 2%, consequently, sequence run times should be no lon-
The mean values of the slopes, intercepts and correlation coeffi- ger than 48 h when analyzing domperidone in the presence of its
cients are given in Table II. process impurities.
Accuracy Conclusion
Accuracy values were determined as percent recovery from the A new rapid UPLC method for the analysis of domperidone, its
linearity samples in the range of 15– 55 mg/mL. Percent recover- impurities and droperidol was successfully developed and vali-
ies for the API ranged from 98.02 to 100.87% and are illustrated in dated on an XDB sub 2 mm column. The analysis time was re-
Table III. duced by 40% from the currently utilized Eur. Pharm. method,
allowing for a much higher sample throughput. Solvent con-
sumption was reduced by 58%, thus greatly reducing solvent
Limit of detection and limit of quantification waste costs and proving the new method to be both economical-
ly and environmentally friendly. Excellent resolution was demon-
The LOD and LOQ were determined experimentally according to
strated throughout the analysis.
the ICH guidelines, following 3 : 1 and 10 : 1 ratios, respectively.
The developed method was validated according to the ICH
The diluted concentrations determined can be seen in Table IV.
guidelines, and the method proved to be accurate, precise, robust
and sensitive. Some stability studies were also carried out; dom-
peridone and its impurities demonstrated no photosensitive re-
Precision actions but were temperature dependent. However, all samples
Precision results were determined as %RSD, which according to remained stable for up to 48 h in ambient temperatures; there-
the ICH guidelines should be ,2%. All three precision studies fore, sequence runs of up to 48 h can now be carried out by
met the acceptance criteria. The results can be seen in Table V. Janssen Pharmaceuticals Ltd without degradation taking place.