177

5.1 Introduction to Spiro alcohol and a brief note on Trospium

chloride

Spiro alcohol, chemically described as 3a-hydroxynortropane-8-spiro-

1’-pyrrolidinium chloride is a key intermediate of Trospium chloride.

Trospium is a quaternary ammonium compound that has antispasmodic,

antimuscarinic effects, but immediately crosses the blood–brain barrier.

Trospium chloride (INN) is a muscarinic antagonist urinary

antispasmodic. It has been developed and patented by Robert Pfleger

(Germany) and is sold under the brand name Sanctura in the US, Tropez

OD in India, Trosec in Canada, Regurin and Flotros in the United

Kingdom and Spasmex in Germany, Russia, Turkey, Argentina, Chile

and Israel. The drug is used for the treatment of overactive bladder.It is

available in Egypt under the brand name Trospikan by Hikma pharma.

SANCTURA®, a muscarinic antagonist, is indicated for the treatment

of overactive bladder with symptoms of urge urinary incontinence,

urgency, and urinary frequency. The recommended dose is 20 mg twice

daily.[1]

An overactive bladder is a condition that results from sudden,

involuntary contraction of the muscle in the wall of the urinary bladder.

Overactive bladder causes a sudden and unstoppable need to urinate

(urinary urgency), even though the bladder may only contain a small

amount of urine.

178

The aim of the present study is to separate and quantify the

impurities in spiro alcohol by a simple and novel technique coupled with

estimation of spiro alcohol in trospium chloride.

The molecular formula of Spiro alcohol is C11H20ClNO and molecular

weight is 217.74

Fig.5.1 Chemical structure of Spiro alcohol

Structure and Chemical name

Molecular weight/Molecular formula

Chemical name: (1R,3r,5S)-3-hydroxyl spiro[bicyclo[3.2.1]octane-8,1'-pyrrolidin]-1'-ium chloride

MolecularFormula: C11H20ClNO Molecular Weight: 217.74

5.2 A Versatile, Validated Method for Separation and Quantification

of Spiro Alcohol and its Related Substances by HPLC-ELSD

5.2.1 Materials and Reagents:

Samples of Spiro alcohol and its three known impurities namely

Impurity-1, Impurity-2 and Impurity-3 [Fig.1] were received from Process

Research Department of Active Pharmaceutical ingredients of Dr. Reddy’s

Laboratories Limited, Hyderabad, India. HPLC grade Formic acid and

Triethylamine were purchased from Merck, Schuchardt OHG, Germany.

179

HPLC grade Acetonitrile was purchased from Rankem, India. High pure

water was prepared by using Millipore Milli Q plus purification system.

5.2.2 Equipment:

The LC system, used for method development, and method validation

was Agilent 1100 series (manufactured by Agilent Technologies, Waldron,

Germany) LC equipped with a ELS detector model: 2000ES and model:

3300 (manufactured by Alltech). The output signal was monitored and

processed using Empower software(Waters) on Pentium computer (Digital

Equipment Co).

5.2.3 Chromatographic conditions:

The chromatographic separation was achieved on an ACE C8

250mm x 4.6mm, 5µm column using a mobile phase containing a

mixture of 0.1% v/v each of triethyl amine and formic acid in water as

solvent-A and further using a mixture of solvent-A and acetonitrile in the

ratio (70:30) (v/v) as solvent-B. The mobile phase was filtered through a

nylon membrane (pore size 0.45µm) and degassed with helium spurge for

5min.The gradient programme : T (min) / % B: 0.01/2, 5/2, 8/50, 15/2

and 20/2 with flow rate of 0.6 mL/min was employed. The HPLC column

was maintained at 25 °C and the detector settings for Alltech 3300 model

are Drift tube temperature: 52°C, Nitrogen gas flow: 1.5 L/min, Gain: 1.

the injection volume was 5µL. Water was used as diluent during the

standard and test samples preparation.

180

A brief synthetic scheme of Trospium chloride is shown in Fig. 5.2.

Fig.5.2 A brief synthetic scheme of Trospium chloride

181

Fig.5.3 Chemical structures of impurities of Spiro alcohol

Impurity name

Structure and Chemical name

Molecular weight/Molecular formula

Impurity-1 (Nortropine)

HN

OH

Chemical name: (1R,3r,5S)-8-azabicyclo[3.2.1]octan-3-ol

MolecularFormula: C7H13NO Molecular Weight: 127.18

Impurity-2 (Tropine)

Chemical name: (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]octan-3-ol

MolecularFormula: C8H15NO Molecular Weight: 141.21

Impurity-3 (Exo isomer)

Chemical name: (1R,3s,5S)-3-hydroxyspiro [bicyclo[3.2.1] octane-8,1'-pyrrolidin]-1'-ium chloride

MolecularFormula: C11H20ClNO Molecular Weight: 217.74

182

5.2.4 Sample preparation:

Standard and test solutions of 10mg/mL were prepared individually

and a stock solution of impurity blend (mixture of Imp-1, Imp-2, Imp-3

and Spiro alcohol) 1000 µg/mL was prepared as well in diluent.

Working solutions of impurity blend (mixture of Impurity-1, Impurity-

2, Impurity-3 and Spiro alcohol) at 2.5µg/mL, 5.0µg/mL, 7.5µg/mL,

10.0µg/mL, 12.5µg/mL and 15.0µg/mL were also prepared in diluent.

Standard and test solutions of 1000 µg/mL were prepared individually

and used for assay determination.

5.3 Method development approach and selection of suitable

chromatographic technique:

Preliminary HPLC experiments were performed by using RI detector

with mobile phase of Triethyl amine and Formic acid buffer mixed with

acetonitrile. All the experiments were relentlessly endured from the

negative peaks near to the analyte peaks, which could lead to

quantification errors during integration. More over, drawback for

gradient mode also restricted to proceed further.

Initial GC experiments discovered that a direct GC measurement with

a flame ionization detector is not practical for the Spiro alcohol, because

of extremely high non-volatility of this quaternary compound. Efforts

were initiated to break the salt selectively to increase volatility by using

quantitative amount of alkaline solution. These experiments endured

from slight degradation of compound which gave inaccurate results. It

183

was established by analyzing spectrally pure compound (about 99% by

NMR) having purity with 92% and two major peaks about 3 to 4%, which

are not reflected in NMR results. This data confirmed the degradation of

compound during the GC analysis. In addition, separation of Exo isomer

is also a limitation, though by varying a range of stationary phases and

temperature programs. Thus the trial prevent a consistent and

simultaneous analysis of Spiro alcohol related substances and assay of

Spiro alcohol. Due to this cause, the choice of GC-FID analysis for this

compound has been proved futile.

To enhance the sensitivity against UV, trials were made to the

selective site pre column derivatization of –OH group in the Spiro alcohol.

Quantification of Spiro alcohol was achieved by derivatisation of analyte

with benoxoprofen reagent was detailed in the literature[2]. In this

method, benoxoprofen reagent reacts with –OH group in analyte to get

fluorescent nature. The procedure involved a number of pre column

derivatisations and solvent extractions especially to increase the

sensitivity and selectivity with poor UV or fluorescent characteristic of

compound. In addition, the pre column derivatisation would limit the

specificity on detection system and they do not authenticate complete

conversion.

Recently successful application of the HPLC coupled with evaporative

light scattering detector (ELSD) would be expected to provide a universal

184

response to all analytes having lower volatility than the mobile pahse

[3].The HPLC-ELSD also present several advantages in comparison to RI

detector in terms of much higher sensitivity for solutes and high baseline

stability even though it is operated under an isocratic or gradient elution

mode [4].Because of the lack of chromophoric group, the use of an ELSD,

therefore, appeared to be a favorable choice for their direct and high

sensitive detection.

In view of the fact that the nebulizing gas flow (N2) rate and

evaporating temperature in an ELSD influence significantly in the rate of

efficiency of response such as detection sensitivity and stability of base

line, initial trail was kept to optimal conditions of the detector settings.

To enhance the sensitivity for ELSD, the flow rate was held as low as

possible at 1.5 L/min, the drift tube temperature at 70°C and gain 1 for

Alltech 3300 model detector.

The major objective is separation of Exo isomer, tropine and

nortropine from Spiro alcohol. Different chromatographic conditions like

different mobile phase compositions, different buffers, various pH and

various diluents were evaluated. Trials were also made by using various

stationary phases like C18, AQ, Phenyl and Cyano. The major problem

found during method development is separation of Exo isomer and early

elution of analyte peaks. Interestingly HCl also provided signals in ELSD,

which were interfering with Exo compound. Finally separation was

185

achieved on C8 column with an isocratic elution of a mixture of Triethyl

amine-Formic acid (0.1% each in aqueous) and Methanol in the ratio of

(95:5) with Alltech 3300 detector.

In the ruggedness study, the same separation was evaluated in

Alltech 2000ES detector by using same conditions. Interestingly, no

signal appeared for nortropine in standard solution. It is due to the

difference in drift tube lengths between two detector models. To a certain

extent, less temperature is required to get the signal for nortropine in

advanced detector. Hence drift tube temperature reduced to 52°C from

70°C, but simultaneously HCl peak response increased drastically and

peak width increased and merged with Exo isomer. To achieve separation

between Exo isomer and HCl, the chromatographic conditions are again

optimized. Finally very good separation was achieved on a C8 column

using a mobile phase containing a mixture of 0.1% v/v each of triethyl

amine and formic acid in water as solvent-A and further using a mixture

of solvent-A and acetonitrile in the ratio (70:30) (v/v) as solvent-B with

gradient elution.

5.3.1 Optimised chromatographic conditions for the determination

of spiro alocohol and its related impurities by ELSD:

Column : ACE C8 250mm*4.6mm*5.0µm

Flow rate : 0.6mL/min

Column temp : 25±2°C

186

Injection volume : 5µL

Diluent : Water

Preparation of Solvent-A: A mixture of 0.1% v/v each of triethyl amine

and formic acid in water

Preparation of Solvent-B: A mixture of Solvent-A and acetonitrile in the

ratio (70:30) (v/v)

Table 5.1: Gradient programme

Time (min) 0.01 5 8 15 20

%Solvent-A 98 98 50 98 98

%Solvent-B 2 2 50 2 2

ELSD Settings for Alltech 3300 model:

Drift tube temperature : 52°C

Nitrogen gas flow : 1.5 L/min

Gain : 1

Fig.5.4 Typical spiked and individual impurities chromatograms

187

188

5.4 Analytical method validation- Results and Discussion:

The method that was developed and optimized in HPLC was

considered for method validation. The analytical method validation was

carried out in accordance with ICH guidelines.[5-6]

5.4.1 System suitability test:

System suitability testing is an integral part of chromatographic

method. The tests are based to ensure that the equipment, analytical

operations, electronics and samples to be analysed make an integral

system and it can be calculated as such.

The Spiro alcohol was spiked with 0.10% exo isomer with respect to

the concentration of Spiro alcohol and injected for three times into

HPLC system. Resolution between Spiro alcohol and exo isomer, tailing

factor for Spiro alcohol was calculated. Good resolution was obtained

between Spiro alcohol and exo isomer [Fig: 5.4]. System suitability results

were tabulated [Table: 5.2].

189

Table 5.2: Results of system suitability

Compound (n=3)

Retention time (Rt)

Resolution between

Spiro alcohol and exo

isomer (Rs)

Tailing factor (T)

Spiro alcohol 4.842 - 0.9

Exo isomer 5.460 1.5 1.7

5.4.2 Limit of Quantification (LOQ) and Limit of Detection (LOD):

LOQ and LOD established for all impurities based on the impurities

dilution linearity method.

5.4.2.1 Methodology for establishment of LOQ and LOD:

The LOQ of an analytical method is the lowest concentration of analyte

in a sample that can be quantitatively determined with appropriate

precision and accuracy. The LOQ is a parameter of quantification used

particularly for the determination of impurities.

LOD and LOQ are determined by injecting linear solutions of all

impurities known concentrations using ten levels ranging from 1.0µg/mL

to 10.0µg/mL. The calculation method is based on the standard

deviation (SD) of the response and the slope (S) of the calibration plot

and using the formula LOQ=10xSD/S and LOD=3.3x SD/S.

190

Table 5.3: LOQ and LOD values of the impurities

S.No. Impurity/product name

LOQ in µg/mL LOD in µg/mL

1 Imp-1 2.2 0.6 2 Imp-2 4.0 1.0 3 Imp-3 2.1 0.5

4 Spiro alcohol 2.0 0.5 5.4.2.2 Precision at Limit of Quantification level:

The precision of related substances method at LOQ level was

determined by injecting six individual preparations of Imp-1, Imp-2 and

Imp-3 with respect to the spiro alcohol concentration. Upon repetitive

injections at quantification limit, the peak properties (retention time,

area) were not influenced. Results have shown negligible variation in

measured responses which revealed that the method was repeatable at

LOQ level with RSD below 2.5%. The % RSD of area of Imp-1, Imp-2 and

Imp-3 for six consecutive injections was 1.6, 2.5 and 1.3 respectively

[Table 5.4].

Table 5.4: Precision results of all impurities at LOQ level

S.No. Imp-1 Imp-2 Imp-3

Prep-1 11254 5025 10458

Prep-2 11298 5002 10535

Prep-3 11087 4987 10458

Prep-4 11025 4835 10369

Prep-5 10924 4935 10258

Prep-6 10845 4702 10654

Average 11072 4914 10455

Stdev 178.92 124.11 135.81

%RSD 1.6 2.5 1.3

191

5.4.2.3 Accuracy at Limit of Quantification level:

Standard addition and recovery experiments were performed to

evaluate accuracy of the developed method for the quantification of all

impurities in spiro alcohol sample at LOQ level.

The recovery study for all impurities was carried out in triplicate at

LOQ level of the target spiro alcohol concentration ( 10 mg/mL). The

method showed constant and high absolute recovery at LOQ level with

a mean absolute recovery of 100.9 %. [Table 5.5]

Table 5.5: Recovery at LOQ level for impurities 1-3

S.No. Impurity name % Recovery

1 Imp-1 100.4

2 Imp-2 100.9

3 Imp-3 101.4

5.4.3 Precision:

The precision of an analytical method convey the closeness of

agreement (degree of scatter) between the series of measurements

acquired from multiple sampling of the same homogeneous sample

under the prescribed conditions. Precision may be measured at three

levels: repeatability, intermediate precision and reproducibility.It is

normally expressed as RSD%.

Repeatability is the results of a method operated over a short interval

of time under the same conditions.

192

Intermediate precision is the end result from within-laboratories

variations due to random events that include different days, different

analysts, different equipment, etc.

Reproducibility is determined by testing the homogeneous samples in

different laboratories. It is a measure of precision between laboratories.

The precision of the related substance method was evaluated by

injecting six individual preparations of Spiro alcohol(10 mg mL-1) spiked

with 0.10% of impurity-1, impurity-2 and impurity-3 with respect to

Spiro alcohol analyte concentration. The % RSD for content of all

impurities for six consecutive determinations was below 0.7[Table 5.6].

Table 5.6: Precision results of the RS method

S.No. Imp-1 Imp-2 Imp-3

1 0.1045 0.1052 0.1038

2 0.1042 0.1055 0.1045

3 0.1047 0.1071 0.1042

4 0.1045 0.1053 0.1049

5 0.1049 0.1059 0.1047

6 0.1059 0.1065 0.1044

Mean 0.1048 0.1059 0.1044

Stdev 0.0006 0.0007 0.0004

%RSD 0.6 0.7 0.4

193

Assay method precision study was evaluated initially by performing

system precision, then by carrying out six independent assays of Spiro

alcohol test sample against qualified reference standard and RSD of six

consecutive assays was 0.1% [Table 5.7]. Results showed insignificant

variation in measured response which demonstrated that the method

was repeatable with RSD below 0.1%.

Table 5.7: Precision results of the assay method

Preparation % Assay

1 99.92

2 99.84

3 99.87

4 99.73

5 99.85

6 99.94

Mean 99.86

Stdev 0.07

%RSD 0.1

Intermediate precision for assay method was performed by carrying

out six independent assays of Spiro alcohol test sample against qualified

reference standard and calculated RSD of six consecutive assays. Related

substances method was also performed by injecting six individual

preparations of Spiro alcohol(10 mg/mL) and 0.10% of impurity-1,

impurity-2 and impurity-3 with respect to Spiro alcohol analyte

194

concentration over different day, different instrument and with different

analyst [Table 5.8].

Table 5.8: Results of Intermediate Precision Study

S.No. Parameter Change

%RSD

for Assay

%RSD for Related Substances

1 Different Analyst (a) Analyst-1 (a) Analyst-2

0.1 0.1

<0.7 <0.4

2 Differnet Instrument

(a) Agilent 1100 series with ELSD model 3000 (b) Agilent 1100 series with ELSD model 2000ES

0.1 0.1

<0.7 <0.6

3

Different Day (a) Day-1 (b) Day-2

0.1 0.1

<0.7 <0.7

5.4.4 Linearity:

5.4.4.1 Linearity of the related substances method:

The linearity of an analytical method is the ability to attain test

results which are directly proportional to the concentration of

analyte with in the given range.

Detector response linearity experiments were carried out by preparing

the Spiro alcohol sample solution containing Imp-1, Imp-2 and Imp-3

covering the range from LOQ–150% (LOQ, 0.025, 0.05, 0.075, 0.10,

195

0.125 and 0.15%) with respect to specification limit (0.10%), was

assessed by injecting seven separately prepared solutions of the normal

test sample concentration (10 mg/mL). The correlation coefficients,

slopes and Y-intercepts of the calibration curve were determined [Table

5.9-5.11].

The Calibration curve was drawn by plotting average area of the

impurity (Imp-1, Imp-2 and Imp-3) on the Y-axis and concentration on

the X-axis [Fig. 5.5] which has shown linear relationship with a

regression coefficient greater than 0.999 for all impurities.

Table 5.9: Linearity of Imp-1

%Conc. w.r.t Spiro alcohol

Concentration in mg/mL Area

LOQ 2.0 5350 25 2.5 6729 50 5.0 14055 75 7.5 20075 100 10.0 27025 125 12.5 34495 150 15.0 41245

correlation coefficient 0.9998

slope 2752.9

y-intercept -151.0

% y-intercept at 100% level -0.56

196

Table 5.10: Linearity of Imp-2

%Conc. w.r.t Spiro alcohol

Concentration in mg/mL Area

LOQ 2.0 11285 25 2.5 14295 50 5.0 28239 75 7.5 40485 100 10.0 54295 125 12.5 68755 150 15.0 83395

correlation coefficient 0.9997

slope 5490.2

y-intercept 218.8

% y-intercept at 100% level 0.40

Table 5.11: Linearity of Imp-3

%Conc. w.r.t Spiro alcohol

Concentration in mg/mL Area

LOQ 2.0 5685 25 2.5 6725 50 5.0 14029 75 7.5 20027 100 10.0 27063 125 12.5 34412 150 15.0 41245

correlation coefficient 0.9997

slope 2738.5

y-intercept -8.6

% y-intercept at 100% level -0.03

197

Fig 5.5: Linearity graph for Imp-1 to Imp-3

5.4.4.2 Linearity of the assay method:

The linearity of the assay method was established by injecting test

sample at the level of 25%, 50%, 75%, 100%, 125% and 150 % of Spiro

alcohol assay concentration (i.e 1000 µg/mL). Each solution was injected

thrice (n=3) into LC system and the average area at each concentration

each calculated. Calibration curve obtained by least square regression

analysis between average peak areas and the concentration shown [Fig.

198

5.6]. A linear relationship with regression coefficient of greater than

0.999. The best fit linear equation obtained was y = 2560x -7739. [Table

5.12]

Table 5.12: Linearity results of the assay method

Concentration (µg/mL) Average Peak area

256.5 653185 503.5 1265845 752.5 1955655 1045.0 2645854 1247.0 3156545 1529.0 3930350

correlation coefficient 0.9998 slope 2560.0

y-intercept -7739.8 % y-intercept at 100% level -0.29

Fig: 5.6: Linearity plot for Assay Method

5.4.5 Accuracy:

The accuracy of an analytical method is measure of the closeness of

test results obtained to the true value.

199

5.4.5.1 Accuracy of the related substances method:

The accuracy of the RS method calculated at 50%, 100% and 150% to

the impurities specification limit (0.10%). The test solution prepared in

triplicate (n=3) with impurities (Imp 1-3) at the level of 0.05%, 0.10% and

0.15% (w.r.t 10 mg/mL test concentration). The mean % recovery of

impurities determined in the spiked test solution by using the area of

impurities in the standard solutions at 0.10% level with respect to Spiro

alcohol analyte concentration [Table 5.13].

Table 5.13: Recovery at 50%, 100% and 150 % level

Impurity Name

Spike level (%)

Added (µg/mL)

Recovered (µg/mL)

Recovery (%)

Imp-1

50 5.02 4.94 98.5

100 10.04 10.28 102.4

150 15.05 14.88 98.9

Imp-2

50 5.03 5.09 101.2

100 10.07 9.96 98.9

150 15.08 15.06 99.9 Imp-3

50 5.09 5.06 99.4

100 10.02 10.01 99.9

150 15.07 15.13 100.4

The related substances method have revealed consistent and high

recoveries at all the three concentration levels i.e 50%, 100% and 150%,

which convey the absolute recovery ranging from 98.5% to 102.4%. The

200

Spiro alcohol recovery study specified that the related substances by LC

method were appropriate for determination/quantification of impurities

in Spiro alcohol.

5.4.5.2 Accuracy of the assay method:

Accuracy of the assay was performed by injecting three preparations

of test sample at the level of 50%, 100% and 150% of analyte (Spiro

alcohol test concentration) i.e 10 mg/mL. The study was performed in

triplicate (n=3), the solution was injected into HPLC system and the

mean peak area of Spiro alcohol analyte peak was calculated for assay

determination. Assay (%w/w) of test solution at each level was

calculated against three injections (n=3) of qualified Spiro alcohol

reference or working standard in terms of Recovered (µg/mL) [Table

5.14].

Table 5.14: Recovery of the assay method for Spiro alcohol

Spike level (%)

Added (µg/mL)

Recovered (µg/mL)

Mean Recovery

(%) %RSD

50 500.35 497.35 99.4

0.5

100 1000.85 984.84 98.4

150 1500.65 1488.64 99.2

The method have shown consistent and high recoveries at all three

concentration (50%, 100% and 150%) levels which mean recovery

ranging from 98.4% to 99.4%. The above accuracy/recovery study

201

indicated that the method was suitable for determination of Spiro

alcohol.

5.4.6 Solution stability:

The solution stability of Spiro alcohol in diluent in the assay method

was performed by leaving the test solutions of sample in tightly capped

volumetric flasks on a laboratory work table with room temperature for

48 hrs. The sample solution was assayed for every six hours interval

upto the study time and freshly prepared reference standard was used

each time to estimate the assay of sample. The %RSD of assay of Spiro

alcohol during solution stability experiments were less than 0.1.

The solution stability of Spiro alcohol in diluent in the related

substances method was carried out by leaving the spiked test solutions

of sample in tightly capped volumetric flasks at bench top, room

temperature for 48hrs (two days). In test spiking solutions, the content of

Imp-1, Imp-2, and Imp-3 are checked for every 12 hours interval up to

the study period. No significant change was observed in the impurity

content during solution stability experiments in the initial values up to

study period. Hence Spiro alcohol spiked sample solution is stable for at

least 48 hrs in the above developed method with the same diluent [Table

5.15- Table5.16].

202

Table 5.15: Summary of related substances content obtained at

different intervals & solution stability

S.No. Interval Imp-1 Imp-2 Imp-3 Total impurities

1 0h 0.22% 0.15% 0.26% 1.11%

2 12h 0.22% 0.16% 0.26% 1.16%

3 24h 0.23% 0.16% 0.27% 1.16%

4 36h 0.23% 0.15% 0.27% 1.14%

5 48h 0.23% 0.16% 0.27% 1.15%

Table 5.16: Summary of assay content obtained at different

intervals & Solution stability

S.No. Interval %Spiro alcohol

1 0h 99.3

2 6h 99.4

3 12h 99.3

4 18h 99.3

5 24h 99.2

6 30h 99.5

7 36h 99.4

8 42h 99.3

9 48h 99.7

Average 99.4

Stdev 0.14

%RSD 0.1

203

5.4.7 Mobile phase stability:

The mobile phase stability of Spiro alcohol in diluent in the assay

method was carried out by fresh test solutions of sample and mobile

phase was kept constant up to 48hrs. The fresh same Spiro alcohol

sample solutions were assayed for every six hours interval upto the study

period, each time freshly prepared reference standard was used to

estimate the assay of sample. The %RSD of assay of Spiro alcohol during

mobile phase stability experiments were less than 0.1.

The mobile phase stability of Spiro alcohol in diluent in the related

substances method was carried out by spiked test solution leaving the

mobile phase at room the temperature for 48 hours. Spiking solution

containing Imp-1, Imp-2, and Imp-3 are checked for every twelve hours

interval up to the study period. No major change was observed in the

impurity content during mobile phase stability study experiments. Hence

Spiro alcohol mobile phase solution is stable for at least 48 hrs in the

above stated analytical method developed [Table 5.17- Table 5.18].

Table 5.17: Summary related substances content obtained at

different intervals & mobile phase stability results of the RS method

S.No. Interval Imp-1 Imp-2 Imp-3 Total impurities

1 0h 0.23% 0.15% 0.25% 1.10%

2 12h 0.21% 0.17% 0.26% 1.15%

3 24h 0.23% 0.15% 0.26% 1.14%

4 36h 0.22% 0.16% 0.25% 1.12%

5 48h 0.25% 0.17% 0.25% 1.16%

204

Table 5.18: Summary assay content obtained at different intervals &

mobile phase stability results of the assay method

S.No. Interval %Spiro alcohol

1 0h 99.2

2 6h 99.3

3 12h 99.0

4 18h 99.1

5 24h 99.4

6 30h 99.0

7 36h 99.4

8 42h 99.2

9 48h 99.2

Average 99.3

Stdev 0.09

%RSD 0.1

5.4.8 Robustness: To determine the robustness of developed method experimental

conditions were intentionally altered and the resolution between critical

pairs and USP tailing factor were evaluated in each deliberately altered

chromatographic conditions [Table 5.19- Table 5.20].

Table 5.19: System suitability-Robustness

Parameters

Conditions

The resolution between the impurity-3 (exo isomer) and TSC

The tailing factor of TSC

Temperature

22ºC 1.4 0.9 25ºC 1.5 0.9 28ºC 1.4 1.0

Different flow

0.5 ml 1.6 1.1 0.6 ml 1.5 0.9 0.7ml 1.4 0.9

205

Table 5.20: Robustness-Relative retention time

Parameters

Conditions

~Relative Retention time Imp-1 Imp-2 Imp-3

Temperature 22ºC 0.58 0.63 0.90

25ºC 0.58 0.64 0.89

28ºC 0.58 0.65 0.92

Different flow 0.5 ml 0.56 0.62 0.88

0.6 ml 0.58 0.64 0.89

0.7ml 0.58 0.65 0.94

5.4.9 Application of the Method to Analysis of Trospium chloride:

As per USP monograph, the content of spiro alcohol in Trospium

chloride is measured by using Thin layer chromatography (TLC). By the

application of this validated method for the quantification of spiro

alcohol, an accurate result can be obtained with in a short run time

when compared to laborious process of TLC.

5.5 Summary and Conclusion:

A simple, stereo selective HPLC-ELSD method developed for the

separation of tropine, nortropine and exo isomer from spiro alcohol and

quantitative determination of spiro alcohol is successfully validated. The

present HPLC-ELSD method could be conveniently applied for the

determination of spiro alcohol in Trospium chloride. [Table 5.21]

206

Table 5.21: Summary of the Validation Results

Test parameter Related substances results Assay method

Imp-1 Imp-2 Imp-3 TSC

Precision

(%RSD) 0.6 0.7 0.4 0.1

LOD (µg/ml) 0.6 1.0 0.5 0.5

LOQ (µg/ml) 2.2 4.0 2.1 2.0

Intermediate precision

0.4 0.3 0.7 0.1

Linearity 0.9998 0.9997 0.9997 0.9998

Accuracy

(% Recovery)

98.5-102.4 95.4-101.2 98.5-100.4 98.4-99.4

Robustness

Rs between

Exo isomer and TSC>1.5

Rs between

Exo isomer and TSC>1.5

Rs between

Exo isomer and TSC>1.5

NA

Solution stability

Stable up to 48hr Stable up to

48hr Stable up to 48hr Stable up to 48hr

Mobile

phase stability Stable up to 48hr

Stable up to 48hr

Stable up to 48hr Stable up to 48hr

REFERENCES:

[1] Product information: SancturaTM, Trospium tablets, Esprit

Pharmaceuticals, East Brunswick, NJ and Indevus Pharmaceuticals,

Inc., Lexington, MA.

[2] Fluorimetric determination of the quaternary compound trospium and

its metabolite in biological material after derivatization with

benoxaprofen chloride Gertrud Schladitz-Keil, Hildegard Spahn, E.

207

Mutschler, Journal of Chromatography B:Biomedical sciences and

applications Volume345 pp 99-110.

[3] Evaporative light scattering detector: a new tool for screening

purposes S.Cardenas, M.Gallego, M.Valcarcel, Anal. Chim. Acta

402(1999) 1-5.

[4] A comparative study of commercial liquid chromatographic detectors

for the analysis of underivatized amino acids Konstantinos Petritis, Claire

Elfakir, Michel Dreux Journal of Chromatography A, 961 (2002) 9–21

[5] International Conference on Harmonization (ICH),. Q2 (R1),

“Validation of Analytical Procedures: Text and methodology” 2005.

[6] Validation of Compendial methods (2007), The United States

Pharmacopeia, 30th edn.USP30-