Vol 4, Issue 3, 2016 ISSN - 2321-4406

Research Article

GREEN VALIDATED METHOD FOR DETERMINATION OF TIEMONIUM METHYL SULFATE

USING REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY TECHNIQUE
WITH STABILITY-INDICATING STUDIES

MAGDA AYAD, MOHAMMED EL-BALKINY, MERVAT HOSNY*, YOUSTINA METIAS

Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt. Email: [email protected]
Received: 14 April 2016, Revised and Accepted: 19 April 2016

ABSTRACT

Objective: The objective of this method was to develop a simple, sensitive, rapid reversed-phase high-performance liquid chromatography (RP-HPLC)
and applied for determination of tiemonium methyl sulfate (TIM) in bulk, pharmaceutical formulations with stability-indicating studies.

Methods: The stability-indicating capability of this method had been established by subjected TIM to several stress conditions of acidic, basic,
oxidative, freezing, heating, photolytic, and catalytic degradation.

Results: Good separation between the target analyte and its degradation products without any interference referring to specificity and selectivity
of this method had been reported. Reversed-phase C18 Kinetex® column (100 mm × 4.6 mm I.D., 2.6 µm), isocratic mobile phase composed of an
aqueous solution adjusted to pH 2.3 by 0.1% orthophosphoric acid-acetonitrile (80:20, v/v) at 0.8 ml/minutes flow rate were used. The assay showed
good linearity over the concentration range of 1-25 µg/ml with a correlation of coefficient >0.999 and with a detection limit of 0.249 µg/ml and a
quantitation limit of 0.755 µg/ml. Results of the analysis were validated statistically by recovery studies.

Conclusion: Validated stability-indicating RP-HPLC has been developed for estimation of TIM in its pure and commercial forms in addition to good
separation from its degradation products within reasonable time.

Keywords: Tiemonium methyl sulfate, Reversed-phase high-performance liquid chromatography, Stability indicating.

INTRODUCTION to 1 µg/ml using a much simpler buffer free with relatively low organic
composition of mobile phase that merits this green chromatographic
method for determination of TIM in pure form and in pharmaceutical
preparations with suitable retention time. Furthermore, the presented
method investigated the stability of TIM under more stress testing
than the previous published HPLC method according to International
Conference on Harmonization (ICH)-recommended forced degradation
conditions showing good selectivity for determination of TIM in the
presence of its degradation product.

METHODS

Instrumentation
An agilent 1100 series chromatographic apparatus equipped with
Chemical structure of tiemonium methyl sulfate
G1313A autosampler injector and 100 µg/L volume injection loop. The
mobile phase was degassed using Agilent G1322A vacuum degasser
Tiemonium methyl sulfate or tiemonium metil sulfate (TIM), with G1311A isocratic quaternary pump and solvent cabinets. UV
4-[3-hydroxy-3-phenyl-3-(2-thienyl)propyl]-4-methyl-morpholinium lamp with short wavelength was obtained from Desaga (Waldbronn,
methyl sulfate with molecular formula C19H27NO6S2 = 429.6, is quaternary Germany) and G1314A variable wavelength detector connected to a
ammonium antimuscarinics which is used in the relief of visceral hp computer loaded with Agilent Chemstation Software were used.
spasms [1]. The drug is not included in any official pharmacopeias. Chromatograms were recorded on Agilent integrator. Separation was
From the literature survey, there were few methods had been reported carried out on Kinetex® 2.6 μ C18 100A (100 mm × 4.6 mm I.D., 2.6 µm
for TIM determination using particle-induced X-ray emission [2] particle size) supplied by Jones chromatography heater. Jenway 4330
and ultraviolet (UV) spectroscopic methods [3] for TIM, its dosage conductivity and pH meter were used.
forms and in the presence of its degradation products [4,5]. Reported
method about stability indicating using high-performance liquid Chemicals and reagents
chromatography (HPLC)/high-performance thin layer chromatography HPLC grade acetonitrile (DUKSAN Pure Chemicals), methanol (Lab-
(TLC) techniques [6] had studied the acid degradation of TIM in its pure Scan Analytical Science), and water (TEDIA® high purity solvents) but
powder form, in pharmaceutical dosage form, and in laboratory prepared all other solvents were of analytical grade such as orthophosphoric acid
mixtures containing different percentages of the degradation product. (Merck Co., Germany), hydrochloric acid, sodium hydroxide, and 30%
H2O2 (El-NASR, Cairo, Egypt) were used in this study.
To our knowledge from the literature, till date, very few HPLC
methods had been mentioned, so our study aimed to develop an easy, TIM raw material was generously supplied by Amoun Pharmaceutical
simple, and more sensitive method as quantitation was applied up Industries Co., (Cairo, Egypt). Its purity was found to be 100.217%
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Innovare Journal of Medical Science, Vol 4, Issue 3, 1-9

according to the comparison method and was used as received without conditions, adopting the reported degradation conditions [4] in
further treatment. addition to thermal, frozen, and photolytic stress (sunlight and UV
light) as follows:
Pharmaceutical preparations
The following available commercial formulations are subjected to Degradation in solutions
analytical procedure: Acidic hydrolysis
1. Visceralgine® tablets, Batch No. 0515261 (Sedico Pharmaceutical
To 20 mg of TIM, 20 ml of 5 N HCl was added in round-bottomed flask.
Company, Giza, Egypt) labeled to contain 50 mg TIM per tablet
2. Viscera ® ampoules, Batch No. 150846, 144766 (Amoun
The resultant solution was refluxed with stirring at 80°C for about 2 hrs
Pharmaceutical Industries Co., Cairo, Egypt) labeled to contain
to facilitate acid degradation of TIM and then tested for degradation
5 mg TIM per 2 ml.
occurrence by TLC using methanol:methylene chloride:glacial acetic
acid (8:2:0.2, by volume) [4]. Degraded solution was allowed to cool to
Chromatographic conditions
room temperature, and after neutralized with 5 N NaOH, the volume was
Samples were analyzed using Kinetex® C18 (100 mm × 4.6 mm I.D.,
completed to 50 ml using HPLC water in 50 ml volumetric flask. Aliquot
2.6 µm particle size) column. Elution pumps run isocratic mobile
0.5 ml of degraded sample was transferred to 10 ml volumetric flask and
phase which consisted of acetonitrile and water adjusted pH 2.3 with
diluted with water to yield concentration 20 µg/ml of TIM; then, it was
0.1% orthophosphoric acid (20:80, v/v). The autosampler utilized
finally injected into HPLC and compared with the control sample.
acetonitrile as a rinse solution; injection volume was 20 µl and flow
rate of mobile phase was 0.8 ml/minutes with pressure 200 bar. The
variable wavelength UV-visible detector was set at 225 nm. The column Alkaline hydrolysis
temperature was maintained at 50°C. To 20 mg of TIM, 20 ml of 5 N NaOH was added in round-bottomed flask.
The resultant solution was refluxed with stirring at 80°C for about 3 hrs
General procedures to facilitate basic degradation of TIM. Degraded solution was allowed
Working standard solutions to cool to room temperature, and after neutralized with 5 N HCl, the
procedure was completed as mentioned in acidic hydrolysis.
A standard stock solution of TIM (100 µg/ml) was prepared by
accurately weighted 10 mg of TIM powder into 100 ml volumetric
flasks, 50 ml of HPLC water was added, shaken, and diluted to volume Oxidative hydrolysis
with the same solvent. The working standard solutions were prepared To 20 mg of TIM, 20 ml of 1 ml of 30% H2O2 diluted with water in
by transferring aliquots of stock solution to 10 ml volumetric flasks round-bottomed flask. The resultant solution was refluxed with stirring
which were completed to final volume with the same solvent to obtain at 80°C for about 3 hrs to facilitate oxidative degradation of TIM.
concentrations ranging from 1 to 25 µg/ml. Degraded yellowish solution was allowed to cool to room temperature
and complete procedures of dilution using water to yield concentration
Construction of calibration curves 20 µg/ml of TIM; then, it was finally injected into HPLC and compared
with the control sample.
Aliquots (0.1, 0.15, 0.2, 0.5, 1, 2, 2.5 ml) of standard solutions were
transferred into a series of 10 ml volumetric flasks then completed
to the mark with the same solvent and mixed to give concentration Frozen stress
equivalent to (1, 1.5, 2.5, 10, 15, 20, 25 µg/ml). 20 µl of the Solution of TIM (100 µg/ml) was prepared using water then frozen
previously freshly prepared solutions were injected (triplicate) in the at −10°C for 14 hrs. Frozen solution was allowed to reach to room
chromatographic system and eluted with the mobile phase under the temperature complete procedures of dilution using water to yield
optimum chromatographic conditions and detected at 225 nm. The concentration 20 µg/ml of TIM; then, it was finally injected into HPLC
standard calibration graph was constructed by plotting peak area and compared with the control sample.
versus the corresponding final concentration in µg/ml; then, the linear
regression equation was computed. Degradation in solid form
Thermal stress studies
Procedures for pharmaceutical preparations A thin layer of TIM bulk drug was spread on a petri dish and subjected
Assay of Visceralgine® tablets to heat at 80°C in a dry heat oven for 8 hrs. 10 mg of TIM sample was
About 10 tablets of analyzed drugs were weighed, finely powdered then accurately weighed and prepared for analysis as previously described.
an amount equivalent to 10 mg of TIM was accurately transferred into
a 100 ml volumetric flask, dissolved in 50 ml methanol and the flask Photo stability (sunlight and UV light)
was sonicated for 30 minutes, the volume was completed to the mark Photolytic studies were conducted by exposing thin layer of TIM bulk
with methanol then followed by filtration [6]. The filtrate of TIM was spread on a petri dish to UV under UV cabinet at 25°C for 4 hrs and
evaporated, and residue of the drug was redissolved with 100 ml HPLC another portion of TIM bulk spread between two glass dishes as thin
water. The general procedure was followed, and recovery experiments layer for 3 days to direct sunlight for determination the effect of light
were performed by direct technique as an additional check on the irradiation on the stability of TIM in the solid state. 10 mg degraded
accuracy of the proposed method. yellowish samples were accurately weighed and prepared for analysis
as previously described.
Assay of Viscera® ampoules
About 4 ml of Viscera ampoules equivalent to 10 mg of TIM was RESULTS AND DISCUSSION
transferred into 100 ml volumetric flask and completed to the mark
with HPLC water. The assay was completed as under general procedure, Method development
and recovery experiments were performed as an additional check on Different conditions affecting the chromatographic performance were
the accuracy of the proposed method. optimized to obtain an acceptable chromatographic resolution with
symmetrical sharp peaks and suitable for good separation between the
Procedures for forced degradation analyte and its main degradation products.

To determine selectivity of the analytical method, the stability- Type of column

indicating capability of the method was performed by exposure of Different columns were used for performance investigations, including
the active ingredient to various stresses as acidic, basic, and oxidative Kinetex® C18 100A (100 mm × 4.6 mm I.D., 2.6 µm particle size), Thermo

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Scientific BDS Hypersil® C18 (150 mm × 4.6 mm I.D., 5 µm particle size), Table 1: Analytical performance data and optimum
Agilent HC C18 (150 mm × 4.6 mm I.D., 5 µm particle size), and Thermo chromatographic conditions for HPLC determination of TIM
Electron Corporation Hypersil Gold® (250 mm × 4.6 mm I.D., 5 µm particle
size). The experimental studies revealed that the first column was the Parameter TIM
most suitable as TIM, and its degradation products were separated and
Beer’s law limits µg/ml 1‑25
eluted within reasonable analysis time showing good symmetrical peak Regression equation*
shape and acceptable chromatographic resolution rather than other Intercept (a) 6.439
columns elongated retention time of TIM and gave broad peaks. Slope (b) 36.818
Correlation 0.9999
Mobile phase composition coefficient (r2)
Selection of mobile phase was based on peak parameters (symmetry, Column Kinetex® C18 100A (100 mm×4.6 mm
retention time, and resolution) and ease of preparation. I.D., 2.6 µm particle size)
Mobile phase Acetonitrile ‑ water adjusted pH 2.3
The optimum chromatographic performance was developed using with 0.1% orthophosphoric acid
acetonitrile and water adjusted pH 2.3 with 0.1% orthophosphoric (20:80%)
acid (20:80, v/v) using the minimum quantity of organic solvent that Flow rate 0.8 ml/minutes
merits this green chromatographic method. A buffer free aqueous Detector UV‑detection at 225 nm
phase was used due to buffer salts can precipitate and cause back Injection volume 20 µl
pressure build-up inside the column in addition to the disadvantage Temperature 50°C
of phosphate salts that have limited high organic solubility. For many Retention time 3.374 minutes
liquid chromatography (LC)-UV and LC-mass spectrometry (MS) *A=a+bC, Where, C=Concentration of drug in µg/ml, A=Absorbance.
methods, a low pH is more important than the presence of a true buffer, HPLC: High‑performance liquid chromatography, TIM: Tiemonium methyl
so 0.1% phosphoric (UV) or formic (MS) acid can be used to satisfy sulfate, UV:Ultraviolet
this requirement as at pH 2.3 gave sharp peaks and high efficiency
in our work. Acetonitrile was selected as organic solvent rather than Table 2: System suitability parameters of chromatogram for the
methanol as giving good symmetrical peak. Various mobile phase determination of TIM by HPLC method
compositions were tried in attempts to obtain good resolution of TIM
and its degradants. Upon varying percentages of the organic phase (15- Parameters TIM Reference value
30%) revealed that increasing the percentage of acetonitrile (e.g., 30%) Retention time (tR) 3.374
showed very early eluted analyte peak while decreasing the percentage Number of 4581 >2000 increase with
(e.g., 15%) increase separation time with relatively broad peak. theoretical plates (N) efficiency of separation
Tailing factor (T) 0.67 ≤2 as T=1 for a typical
Column oven temperature symmetric
Column oven temperature was also studied at room temperature Capacity factor (Kˋ) 1.48 1‑10 acceptable
40 and 50°C. It was found that column temperature 50°C was optimum. HETP 0.022 mm The smaller the value, the
higher the column efficacy
Choice of detection wavelength HETP: Height equivalent to one theoretical plate, TIM: Tiemonium methyl
TIM showed main absorption peaks at 225, 235, and 250 nm. 225 nm sulfate, HPLC: High‑performance liquid chromatography
was found to be optimum for detection at which the highest detector
response was obtained.
Method validation
The developed method was validated according to the ICH guidelines [9]
Choice of flow rate
by testing the followings:
The effect of flow rate was studied to optimize the chromatographic
efficiency of the proposed method and improve the resolution of the
eluted peaks. Linearity
Calibration curve was constructed, for determination of TIM by
The flow rate was changed over the range of 0.7-1 ml/minutes and the proposed method, by plotting peak area responses against
a flow rate of 0.8 ml/minutes was optimum for good separation in a concentrations of the drug in a range of 1-25 µg/ml to give a rectilinear
reasonable time. graph. The linear regression equation was derived using the least-
squares method, and statistical analysis of data for the drug was listed in
Hence, the optimum chromatographic performances were achieved as Table 5 proving the good linearity over the working concentration range.
summarized in Table 1 for quantification of the drug and separation
from its degradation products without interference from each other
Sensitivity
within 8 minutes.
In accordance with ICH guideline Q2(R1) [9], several approaches
Satisfactory symmetrical peak shape (Fig. 1), number of theoretical to determine the detection and quantitation limits include visual
plates (N), tailing factor (T), and capacity factor (k´) and their reference evaluation, signal-to-noise ratio, and the use of standard deviation of
values [7,8] are shown in Table 2. the response and the slope of the calibration curve. In the present study,
the limit of detection (LOD) and limit of quantitation (LOQ) were based
Stability studies on the third approach and were evaluated using the following equation:
Results of degradation studies indicated that TIM underwent completely
σ
degradation in acid (heating for long time or at higher temperature under LOD = 3.3
this acidic stress cause formation of small black particles), extensive s
oxidative degradation, moderate degradation occurred in alkaline σ
medium and sunlight degradation while relatively stable in thermal, LOQ = 10
s
frozen, and UV light conditions as summarized in Tables 3 and 4.
Where, σ = The standard deviation of replicate blank responses (under
Figs. 2-8 showed chromatograms obtained from TIM after forced the same conditions as for sample analysis), and S = The slope of the
degradation under several stress conditions. calibration curve of the analyte.

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Fig. 1: Representative chromatogram of 25 µg/ml tiemonium methyl sulfate under the described conditions

Table 3: Results from analysis of samples after forced degradation study

Stress condition Time and Retention time of Recovery of Remarks

temperature degradants (minutes) intact drug (%)
Acid degradation Reflux 2 hrs at 80°C 6.343 and 7.068 0 Completely degradation was observed at
5 N HCL this two peaks
Alkaline degradation Reflux 3 hrs at 80°C 6.540 84.1 One small peak
5 N NaOH
Oxidative degradation Reflux 3 hrs at 80°C 2.802, 4.800 and 5.963 28.94 Small three peaks
30% H2O2
Frozen stress 14 hrs at 10°C 96 Relatively stable as no degradation product
was observed
Thermal stress dry 8 hrs at 80°C 97.60 Relatively stable as no degradation product
heat oven was observed
Sunlight degradation 3 days 3.987 87.82 Physical change in powder color (yellow)
Small peak appears only when change
wave length at 325 nm
UV light degradation For 4 hrs at 25°C 4.057 and 4.781 96.51 Physical change in powder color (yellow)
Two small peaks
UV: Ultraviolet

Table 4: System suitability parameters of degradation products of TIM

Degradation medium Degradation product Retention time (minutes) Capacity factor (K´) Selectivity (α) Resolution (Rs)
Acidic A1 6.343 3.879 1.84 9.00
A2 7.068 4.437 1.11 1.68
Alkaline B1 6.540 4.030 1.91 10.35
Oxidative O1 2.802 1.155 ‑ ‑
O2 4.800 2.692 1.39 5.53
O3 5.963 3.587 1.24 3.89
Sun light S1 3.987 1.492 1.33 2.311
UV light U1 2.075 0.596 ‑ ‑
U2 2.265 0.742 1.09 1.22
U3 4.057 2.12 1.19 2.82
U4 4.781 2.678 1.18 3.02
TIM: Tiemonium methyl sulfate, UV: Ultraviolet

Accuracy and precision method. Statistical comparison of the results was performed using
Accuracy Student’s t-test and variance ratio F-test at 95% confidence level
The accuracy of the proposed method was ascertained by (Table 6).
determining pure samples of the cited drugs with reported method. Precision
Statistical analysis [10] of the results obtained by the proposed • Intra-day precision: To determine intra-day precision (repeatability)
and comparison method [3] showed that the calculated values of the proposed methods, solutions containing three different
did not exceed the theoretical ones which indicated no significant concentrations (within working ranges) of each drug in its pure
differences found between the proposed method and comparison form were prepared and analyzed by proposed methods on three

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Fig. 2: Acid degradation chromatogram of 20 µg/ml tiemonium methyl sulfate

Fig. 3: Alkaline degradation chromatogram of 20 µg/ml tiemonium methyl sulfate

Fig. 4: Oxidative degradation chromatogram of 20 µg/ml tiemonium methyl sulfate

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Fig. 5: Chromatogram of 20 µg/ml tiemonium methyl sulfate under frozen stress

Fig. 6: Chromatogram of 20 µg/ml tiemonium methyl sulfate under thermal stress

Fig. 7: Photodegradation chromatogram of 20 µg/ml tiemonium methyl sulfate using ultraviolet light

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a b
Fig. 8: (a) Photodegradation chromatogram of 20 µg/ml tiemonium methyl sulfate (TIM) under sunlight at 225 nm, (b) photodegradation
chromatogram of 20 µg/ml TIM under sunlight at 325 nm

Table 5: Assay results for the determination of TIM in pure form (within working ranges) of the cited drugs were carried out using
by the proposed method proposed method over period of 3-day.

Parameters TIM Intra- and inter-day precisions and accuracy results were summarized
in Table 7 indicating the validity, applicability of the proposed methods,
Taken µg/ml Found µg/ml Recovery*%
and the reproducibility of the results.
1 0.985 98.49
1.5 1.482 98.79 Specificity
2 1.971 98.54 Specificity of the proposed method allowed separation and
5 4.986 99.71 determination of TIM in its dosage forms as tablet, ampoule and in the
10 10.149 101.49 presence of its degradation products without any interference.
15 14.989 99.93
20 19.924 99.62
Robustness
25 25.015 100.06
Mean±SD 99.58±0.994 Robustness is the measure of the capacity of proposed methods to
N 8 remain unaffected by small variations of the method variables. It was
RSD 0.998 evaluated by making small incremental changes in one parameter
SE 0.351 while the others were kept unchanged as flow rate (0.77, 0.8 and
Variance 0.988 0.83 ml/minutes) and injected volume (19.5, 20, and 20.5 µl). The
Slope 36.818 effect of the changes was studied on the peak area and retention
LOD (µg/ml) 0.249 time by calculating (recovery ± % RSD) as shown in Table 8, and this
LOQ (µg/ml) 0.755 changes had negligible influence on the shape of the peak and tailing
ε (×104)** 1649.46 factor.
L.mol−1.cm−1
*Average of three different determinations, **Apparent molar absorptivity was System suitability
calculated on the basis of the molecular weight of the drug. TIM: Tiemonium
methyl sulfate, SD: Standard deviation, RSD: Relative standard deviation, System suitability test parameters: Column efficiency number of
SE: Standard error, LOD: Limit of detection, LOQ: Limit of quantitation theoretical plates (N), capacity factor (K´), tailing factor (T), resolution
(Rs), and selectivity (ɑ) are used to verify that the resolution and the
reproducibility of the chromatographic system are adequate for the
Table 6: Statistical analysis of results obtained by the proposed
analysis to be done according to United States Pharmacopeia [11] as
method compared with reported method shown in Tables 2 and 4.
Drug Proposed Comparison Analytical applications
methods method
Dosage form analysis
TIM
Mean±SD 99.58±0.994 100.22±0.964 The proposed method was successfully applied to the assay of the
Variance 0.988 0.930 studied drugs in their pharmaceutical formulations Visceralgine® tablets
N 8 6 and Viscera® ampoules. Satisfactory results obtained for the recoveries
Student’s t‑test 1.207 (2.179)* of the drugs were in good agreement with the label claim and proved
F‑test 1.062 (3.97)* the suitability of the proposed methods. The percentage recoveries of
*The corresponding theoretical values for t‑ and F‑tests at p=0.05. SD: Standard the pure drugs using the proposed methods shown in Table 9 are in
deviation, TIM: Tiemonium methyl sulfate good agreement with those obtained with the comparison methods [3].
Fig. 9 show representative chromatograms for determination of TIM in
its dosage forms.
successive times in the same day. The values of relative standard
deviation (RSD) were calculated, and percentages relative error CONCLUSION
(Er%) of the suggested method were also calculated using the
following equation: Validated stability-indicating reversed-phase HPLC has been developed
Er% = [(found − added)/added] × 100 for estimation of TIM in its pure and commercial forms in addition to
• Inter-day precision: To establish inter-day precision (intermediate), good separation from its degradation products within a reasonable time.
three experimental replicates including three different concentrations It has the advantage of being much simpler, and friendly environmental

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Table 7: Precision data for the determination of TIM by the proposed method

TIM
Intra‑day Inter‑day
Added (µg/ml) Found±SE (µg/ml) RSD% Er% Added (µg/ml) Found±SE (µg/ml) RSD% Er%
5 5.14±0.526 0.886 2.74 5 5.01±0.608 1.052 0.14
15 15.08±0.299 0.516 0.51 15 14.97±0.573 0.994 −0.18
25 24.86±0.313 0.545 −0.57 20 20.03±0.612 1.058 0.16
TIM: Tiemonium methyl sulfate, SE: Standard error, RSD: Relative standard deviation

Table 8: Robustness of the proposed method

Concentration Variation Retention time Peak area Tailing factor Theoretical plates
5 µg Flow (0.77 ml/minutes) 3.543 183.102 0.77 4723
Flow (0.83 ml/minutes) 3.266 182.641 0.81 5184
Without variations (0.8 ml/minutes) 3.376 186.018 0.7 4655
RSD of affected parameters 4.108 1.032
10 µg Injected volume (20.5 µl) 3.392 392.105 0.68 4438
Injected volume (19.5 µl) 3.369 368.671 0.68 4675
Without variations (20 µl) 3.354 379.277 0.69 4614
RSD of affected parameters 0.568 3.141
RSD: Relative standard deviation

Table 9: Application of the proposed method for determination of TIM in its dosage forms

Parameters TIM
Visceralgine® tablets Viscera® ampoules
Taken µg/ml Added µg/ml Recovery* % Taken µg/ml Added µg/ml Recovery* %
3 2.921 97.38 6 6.118 101.97
4 3.954 98.84 7 7.105 101.50
5 4.847 96.94 8 8.157 101.96
6 5.823 97.05 10 10.126 101.26
Mean±SD 97.55±0.878 101.67±0.350
N 4 4
SE 0.439 0.175
Variance 0.771 0.122
*Mean of three different experiments. SE: Standard error, SD: Standard deviation, TIM: Tiemonium methyl sulfate

a b
Fig. 9: Chromatogram of tiemonium methyl sulfate sample solutions from (a) The tablets (6 µg/ml), (b) the ampoules (7 µg/ml)

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