Corresponding author: Anas Alshishani (
Sensitive, simple, and fast LC-MS/MS method for the determination of Scopolamine in human plasma was developed and validated. Liquid-Liquid extraction technique was used for sample preparation. Cyano bonded phase column (150 × 4.6 mm, 5 µm) was used for the separation with an isocratic elution of ammonium format buffer:methanol (60:40) mobile phase at a flow rate of 1 ml.min-1 over 3.8 min run time. Scopolamine and [13C,2H3]-Scopolamine, as internal standard, were detected and quantified in positive ion mode via
Scopolamine, also known as hyoscine, is a natural alkaloid drug obtained from several plants such as
Chemical structure of Scopolamine.
Many analytical methods had been developed and validated to quantify Scopolamine in human plasma as a single compound or simultaneously with other alkaloids. Scopolamine in urine and plasma was measured using a semi-automated solid-phase extraction followed by a radioreceptor test (
In this work, an LC-MS/MS method has been developed and fully validated for the determination of Scopolamine in human plasma using K3EDTA anticoagulant. The method was sensitive and robust, yet it was simple to be applied in any ordinary bioanalytical laboratory. The method was successfully applied to a bioequivalence study of scopolamine transdermal patch formulations involving 16 healthy Jordanian volunteers.
Scopolamine hydrobromide and internal standard (
Chromatographic separation was achieved using a Shimadzu Nexera XR system (Kyoto, Japan) using cyano column (150 × 4.6 mm, 5 µm) obtained from ACE (Reading, UK). The auto-sampler temperature was 6 °C. The analyte and
Standard solutions of scopolamine were prepared from stock solution (160 µg.ml-1) and
For extracting Scopolamine and
The developed method was validated according to the in the FDA and EMA guidelines.Selectivity, sensitivity, linearity, matrix effect, precision, accuracy, integrity, stability, and dilution recovery were all evaluated. The selectivity was evaluated by injection of eight different lots of hemolyzed and hyperlipidemic blank plasma. Caffeine, Paracetamol, Diclofenac, ascorbic acid, Nicotine, Aspirin, and Ibuprofen were all examined as potential concomitant medication interference. The standard calibration curves were evaluated by plotting eight different levels.Sensitivity was tested by analyzing six triplicates of
The pharmacokinetic parameters of Scopolamine were measured in sixteen healthy Jordanian volunteers in a by measuring the rate and extent of scopolamine after using scopolamine transdermal patch. This study was approved by the Institutional Review Board/Independent Ethics Committee (
Both positive and negative ionization modes were investigated for Scopolamine. The positive mode response was more suitable than negative mode. Chromatographic parameters were optimized to achieve high resolution and improved scopolamine signal intensity yet maintaining a short run time. Scopolamine detection was enhanced after addition of formic acid to the mobile phase. Different ratios of mobile phase were evaluated, and the optimum ratio was 60:40 of ammonium format buffer and methanol. Many columns stationary phases and brands were tested and ACE Cyano (150 × 4.6 mm, 5 µm) was the optimum one and was used for chromatographic separation. The retention time of Scopolamine and
Selectivity reflects the degree of interference of Scopolamine and
The sensitivity of the developed method was determined using
Matrix effect quantitative evaluation of Scopolamine and
Over the concentration range of 3.03–315.76 pg.ml-1 the method was found liner. A regression equation with a weighting factor (1/x2) of Scopolamine to the
The accuracy of the developed method was evaluated in terms of % Recovery, and it was found to be ranged from 96.89 to 107.57% % for intra-day accuracy and from 97.74 to 110.53% % for inter-day accuracy. The inter- and intra-day precision were evaluated using 18 replicates and six replicates, respectively. The CV% for both precisions was in the range of 1.28 to 10.46%. The results summarized in Table
Intra-day and inter-day precision and accuracy for scopolamine.
Intra-day (n = 18) | Inter-day (n = 6) | |||||||
---|---|---|---|---|---|---|---|---|
Measured concentration (pg/mL) | Mean concentration found (pg/mL) | Accuracy (%) | CV (%) | Measured concentration (pg/mL) | Mean concentration found (pg/mL) | Accuracy (%) | CV (%) | |
|
3.03 | 3.10 | 102.31 | 10.12 | 3.03 | 3.18 | 104.95 | 10.46 |
|
9.09 | 9.78 | 107.59 | 4.97 | 9.09 | 10.01 | 110.12 | 2.74 |
|
60.63 | 65.08 | 107.34 | 3.87 | 60.63 | 67.01 | 110.53 | 1.55 |
|
126.30 | 122.38 | 96.89 | 2.18 | 126.30 | 123.45 | 97.74 | 1.39 |
|
236.82 | 235.64 | 99.50 | 2.64 | 236.82 | 232.39 | 98.13 | 1.28 |
Liquid-Liquid extraction technique with Ethyl acetate and n-Hexane (70:30) was robust, effective, and simple. The percent recovery was assessed by comparing the peak area ratio of scopolamine in the treated samples with those in the un-treated samples, as showed by the following equation:
The recoveries of scopolamine and
The dilution integrity was assessed for samples whose concentration was as double as ULOQ concentration, which were named as
The stability test shows that scopolamine was stable in human plasma at 25 °C for 18 h. The result of the extracted plasma samples indicates that samples were stable in the auto-sampler (25 °C) for 43 h, and they were stable after subjected to four freeze and thaw cycles.
Stability data for scopolamine in human plasma samples (n = 6).
Stability test | Mean ± SD (pg/mL) | Accuracy /Stability (%) | Precision (%) | |
---|---|---|---|---|
Bench top stability | 9.09 | 9.85 ± 0.42 | 108.32 | 4.25 |
(25 °C for 18 h) | 236.82 | 235.17 ± 2.06 | 99.30 | 0.88 |
Injection phase stability | 9.09 | 8.92 ± 0.29 | 102.85 | 4.94 |
(2–8 °C for 43 h) | 236.82 | 232.33 ± 4.44 | 100.76 | 1.57 |
Autosampler stability | 9.09 | 9.35 ± 0.46 | 98.11 | 3.19 |
(25 °C for 43 h) | 236.82 | 238.62 ± 3.74 | 98.11 | 1.91 |
Freeze-thaw stability | 9.09 | 9.86 ± 0.20 | 108.43 | 2.04 |
(four cycles) | 236.82 | 235.02 ± 3.07 | 99.24 | 1.30 |
Long-term stability | 9.09 | 9.80 ± 0.24 | 107.71 | 2.46 |
(-70 °C for 3 day) | 236.82 | 227.93 ± 3.08 | 96.94 | 1.35 |
The suggested and validated LC-MS/MS method has been successfully implemented for measuring the pharmacokinetic parameters of scopolamine in 16 healthy male volunteers. Scopolamine concentration/time profiles from all volunteers after receiving Scopolamine transdermal patch (1 mg/3 days) are presented in Fig.
Mean plasma concentration following administration of a single transdermal patch of Scopolamine (1 mg/ 3 days).
The results of the pharmacokinetic parameters illustrated that the average maximum plasma concentration (Cmax) of scopolamine for the twenty subjects was 95.787 ± 36.877 pg.mL-1 and reached at the average time of 36.03 ± 21.86 h. The other parameters were the area under the curve (AUC0-t and AUC0-∞) for scopolamine and those were found to be 1945.255 ± 788.994 pg.h.ml-1 and 5425.862 ± 1259.914 pg.h.ml-1 for AUC0-t and AUC0-∞, respectively as illustrated in Table
Pharmacokinetic parameters of Scopolamine transdermal patch (1 mg/3 days) (Mean ± SD).
Parameters | Scopolamine |
---|---|
Cmax ± SD (ng.ml-1) | 95.787 ± 36.877 |
Tmax (h) | 36.03 ± 21.86 |
T1/2 (h) | 6.553 ± 2.690 |
AUC0-t (ng.h.ml-1) | 1945.255 ± 788.994 |
AUC0-∞ (ng.h.ml-1) | 5425.862 ± 1259.914 |
Kel (h-1) | 0.0498 ± 0.0137 |
A rapid, simple, and sensitive LC-MS/MS for scopolamine quantification in human plasma was developed and fully validated according to FDA and EMA guideline. The used liquid-liquid extraction technique gave consistent and reproducible recoveries for Scopolamine. The method was accurate and precise for the determination of scopolamine in human plasma throughout a concentration range of 3.03–315.76 pg.ml-1 and should be useful for regular monitoring of drug concentrations in pharmacokinetic investigations. The method was successfully applied to determine scopolamine transdermal patches in healthy subjects and pharmacokinetic parameters were calculated.
The authors would like to thank the deanship of scientific research at the Zarqa University for the financial support. Also, thanks to Pharmaceutical Research Unite (