STRUCTURE OF CHEMICAL COMPOUNDS,

METHODS OF ANALYSIS AND PROCESS CONTROL

DEVELOPMENT AND VALIDATION OF A METHOD

FOR QUANTITATIVE DETERMINATION OF KEMANTANE DRUG

SUBSTANCE BY GAS CHROMATOGRAPHY

A. V. Tolkacheva,1,2 L. N. Grushevskaya,1 N. I. Avdyunina,1 B. M. Pyatin,1

V. I. Prokof’eva,2 and L. M. Gaevaya1

Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 47, No. 12, pp. 42 – 47, December, 2013.

Original article submitted October 29, 2013.

A new technique for quantitative determination of the active ingredient in pharmaceutical drug substance

kemantane (5-hydroxyadamantan-2-one) using gas chromatography with a flame-ionization detector

(GC-FID) was developed and validated. The chromatographic separation was performed on a WCOT fused

silica capillary column (CP-WAX 52 CB, 50 m � 0.32 mm, 1.2-mm-thick layer of stationary phase). The opti-

mum parameters of the chromatographic system and robustness factors of the technique were determined. The

technique was validated with respect to the main parameters. Several batches of pharmaceutical kemantane

drug substance were analyzed.

Keywords: kemantane, 5-hydroxyadamantan-2-one, pharmaceutical drug substance, quantitative determina-

tion of active ingredient, GC-FID, GC chromatography, adamantane derivatives, validation, oximation, titra-

tion in non-aqueous medium.

Kemantane (I), 5-hydroxyadamantan-2-one, is a struc-

tural derivative of adamantane. Studies of the pharmacologi-

cal activity of I at Zakusov State Institute of Pharmacology,

Russian Academy of Medical Sciences (ZSIP RAMS),

showed that it possesses anti-Parkinson’s and immunotropic

activity and is capable of relieving alcohol abstinence syn-

drome. Also, it has high cerebrovascular activity and en-

hances blood flow to ischemic tissue [1, 2]. The high phar-

macological activity of I indicated that further research on

the pharmaceutical development of I as a drug substance was

advisable.

A method for quantitative determination of I drug sub-

stance was proposed earlier. It consisted of oximation of the

ketone by hydroxylamine hydrochloride in the presence of an

EtOH solution of N,N-diphenylguanidine, a strong base that

removed HCl from hydroxylamine hydrochloride [3]. The

excess of hydroxylamine was titrated with an EtOH solution

of HClO4

(0.2 M) in the presence of bromophenol blue indi-

cator. The color of the indicator near the equivalence point

was compared with that of a blank sample consisting of a so-

lution of hydroxylamine hydrochloride and EtOH in order to

increase the accuracy of the end-point determination during

titration of the test sample. It was necessary to observe rigor-

ously the order of addition of the components because of the

danger of forming acetals and ketals. A control test was car-

ried out in parallel. Later, changes in the ratios of used re-

agents were introduced and potentiometry was proposed for

determining the titration end-point.

The oximation method gave reproducible results if the

aforementioned conditions were rigorously observed. How-

ever, it had serious drawbacks such as long reagent prepara-

tion times, complications in determining the equivalence

point, and the presence in I of impurities containing ketones

and reacting with hydroxylamine under the titration condi-

tions.

664

0091-150X/14/4712-0664 © 2014 Springer Science+Business Media New York

Pharmaceutical Chemistry Journal, Vol. 47, No. 12, March, 2014 (Russian Original Vol. 47, No. 12, December, 2013)

1Zakusov State Institute of Pharmacology, Russian Academy of Medical

Sciences, Moscow, 125315 Russia.2

Sechenov First Moscow State Medical University, Moscow, 119991 Russia

The goal of the present work was to find a more specific,

accurate, and reproducible method for quantitative determi-

nation of I drug substance. For this, we developed a tech-

nique for quantitative determination of I using gas chroma-

tography (GC) and also compared the drug substance analyt-

ical results obtained by the GC technique with those from

oximation.

EXPERIMENTAL PART

We studied samples of I drug substance and its technical

impurities adamantan-2-one (IV) and adamantane-2,6-dione

(II), which were prepared by the Drug Synthesis Technology

Group at ZSIP RAMS. 1-Adamantaneethanol (III) (Aldrich

Chem. Co., CAS No. 6240-11-5) was used as an internal

standard for developing the technique.

I drug substance is characterized visually as a white or

almost white crystalline powder. It is readily soluble in H2O,

EtOH (95%), and CHCl3

and difficultly soluble in hexane

and petroleum ether. The technical impurities (II and IV) are

white crystalline compounds that are readily soluble in

MeOH and CHCl3, soluble in EtOH and H

2O, and moder-

ately soluble in hexane. The internal standard III is a white

crystalline powder with a cream tint that is poorly soluble in

H2O and readily soluble in EtOH.

Compound I was analyzed quantitatively by oximation

using the following procedure. The drug (~0.15 g, accurate

weight) was dissolved in hydroxylamine hydrochloride solu-

tion (8 mL), treated accurately with diphenylguanidine solu-

tion (0.05 M, 40 mL), stirred, left to stand protected from

light for 30 min, and titrated with alcoholic HClO4

(0.2 M).

The titration end-point was determined by potentiometry.

A control test was carried out in parallel.

One milliliter of alcoholic HClO4

(0.2 M) corresponded

to 0.03324 g of C10

H14

O2, which should be at least 98.5%

and less than 101.0% calculated as the dry substance.

Preparation of hydroxylamine hydrochloride solu-

tion. Hydroxylamine hydrochloride (7.0 g) was dissolved in

H2O (20 mL) in a 200-mL volumetric flask. The solution

volume was adjusted to the mark using EtOH (95%).

Preparation of diphenylguanidine solution (0.05 M).

Diphenylguanidine (5.3 g, pure, MRTU 6-09-4811–67) was

dissolved in EtOH (95%) (200 mL) in a 500-mL volumetric

flask. The solution volume was adjusted to the mark with the

same solvent and stirred.

Preparation of bromophenol blue solution. Bromophenol

blue (1 g) was dissolved in EtOH (95%) (100 mL).

Preparation of alcoholic HClO4

(0.2 M). HClO4

solution

(20.52 mL, 65%) was placed into a 1-L volumetric flask, ad-

justed to the mark with EtOH (95%), and stirred.

HClO4

solution titration. Diphenylguanidine (~0.4 g, ac-

curate weight, pure, MRTU 6-09-4811–67) was dried at

105°C to constant weight, dissolved in anhydrous EtOH

(25 mL), and titrated with the prepared HClO4

solution in the

presence of one drop of bromophenol blue solution until the

violet color turned yellow.

The calibration coefficient was calculated using the first

method, where T = 0.04276.

The potentiometric titration end-point was determined on

a SevenEasy S20-K instrument (Mettler Toledo) with an

Inlab 413 universal electrode.

An alternative technique for quantitative determination

of the active ingredient in kemantane drug substance was de-

veloped and validated on a GC with a flame-ionization de-

tector (Varian GC-450, Netherlands) and an autosampler

(Varian PAL Autosampler, Netherlands). The signal and ob-

Development and Validation of a Method for Quantitative Determination 665

O

O

O

O

OH

OH

IV

III

II

I

Fig. 1. Typical chromatogram of model mixture (concentration of each compound 0.5 mg/mL): kemantane (I), adamantane-2,6-dione (II),

1-adamantaneethanol (III), adamantan-2-one (IV).

tained results were processed using the Galaxie program

(Galaxie Chromatography Data System, version

1.9.302.952).

The technique for quantitative determination of I in the

drug substance by GC was developed using samples of I

drug substance with various contents of technical impurities.

Both nonpolar (hexane, EtOAc) and weakly polar sol-

vents (MeOH, EtOH, H2O) were examined as the solvent for

sample preparation. EtOH (95%) was used as the solvent be-

cause of the solubility of the analyzed compounds (I, III, and

possible accompanying technical impurities II and IV) and

also the influence of the solvent on the chromatographic sep-

aration.

RESULTS AND DISCUSSION

The following conditions were chosen as a result of the

studies: fused silica capillary column CP-WAX 52 CB

(50 m � 0.32 mm) with polyethyleneglycol stationary phase

(1.2 �m); vaporizer temperature 220°C; thermostat tempera-

ture 200°C; detector temperature 230°C; carrier gas (N2)

flow rate 5 mL/min; purge gas (N2) flow rate 25 mL/min; H

2

flow rate 30 mL/min; air flow rate 300 mL/min; flow divi-

sion 1:40.

Under these conditions, I and its technical impurities

were completely separated (Fig. 1).

The internal standard method was proposed for quantita-

tive determination of I in the drug substance.

The choice of standard was based on satisfying the fol-

lowing requirements. It should have a chemical structure

similar to that of the determined compound. It should appear

in the chromatogram as a well resolved peak.

Compound III was chosen as the internal standard be-

cause it structure was similar to that of the determined com-

666 A. V. Tolkacheva et al.

0 0.2 0.4 0.6 0.8 1.0 1.2

4000

3000

2000

1000

0

1

2

y x= 4343.5 – 0.8812

y x= 3513.2 – 11.747

R2

= 0.9999

R2

= 0.9999

S, mV · min

C, mg/mL

Fig. 2. Peak area as a function of concentration: 1-adamantane-

ethanol (III) (1 ) and kemantane (I) (2 ).

TABLE 1. Separation Parameters of Model Mixture

Compound RT As Rs

Adamantan-2-one 5.26 1.08 –

1-Adamantaneethanol 12.04 1.03 31.02

Adamantane-2,6-dione 18.60 1.00 14.31

Kemantane 26.02 1.04 17.96

1

2

Fig. 3. Typical chromatogram of model mixture: 1-adamantaneethanol (III) (1 ) and kemantane (I) (2 ) at concentration 0.5 mg/mL.

pound. Also, its peak was completely separated from those

of I, the technical impurities in I, and the solvent peak under

the given chromatography conditions.

Figure 1 shows a typical chromatogram of the model

mixture of I, its technical impurities, and the internal stan-

dard (III). Table 1 lists the separation parameters [retention

time (RT, min), asymmetry factor (As), and separation be-

tween two neighboring peaks (Rs)].

A study of the effect of changing the chromatography

conditions on the separation parameters of kemantane, the

technical impurities, and the internal standard showed that

varying the temperature by ±10°C changed the kemantane

peak shape. The asymmetry coefficient at 200°C was 1.40; at

190°C, 1.47; and at 210°C, 2.75. A change of 5°C had an in-

significant effect on the kemantane peak symmetry. The

asymmetry coefficient increased to 1.33 – 1.35.

Changing the carrier gas flow rate by ±1 mL/min af-

fected only the retention time of the compound peak without

affecting its shape.

Eliminating or reducing the flow division overloaded the

column with solvent (EtOH) and decreased its efficiency

whereas increasing the flow division decreased the sensitiv-

ity of the technique. The optimum combination of column ef-

ficiency and technique sensitivity was observed for flow di-

vision 1:40.

The limits of linearity of the peak areas as a function of

solution concentrations of the studied compounds were de-

termined in order to choose the working concentrations of

solutions of I and III. The functions for both I and III were

linear in the range from 0.01 to 1 mg/mL. The correlation co-

efficients were 0.9999 (Fig. 2).

The detection limit of I was 0.00025 �g; of III,

0.000125 �g. The limit of quantitation of I was 0.0005 �g; of

III, 0.00025 �g.

Based on the results, we set the working concentration

for solutions of I and III at 0.5 mg/mL.

Figure 3 shows a typical chromatogram of a model mix-

ture of I and III with the concentrations of each at

0.5 mg/mL.

The following technique was developed based on the re-

sults. I drug substance (~0.025 g, accurate weight) was

placed into a 25-mL volumetric flask and dissolved in EtOH

(95%, 10 mL). The volume was adjusted to the mark with the

same solvent. The mixture was stirred. The resulting solution

(5 mL) was placed into a 10-mL volumetric flask into which

internal standard solution (5 mL) was placed beforehand and

mixed thoroughly (test solution, concentration of I

0.5 mg/mL).

Preparation of internal standard (III) solution. Com-

pound III (~0.025 g, accurate weight) was placed into a

25-mL volumetric flask and dissolved in EtOH (95%,

10 mL). The volume was adjusted to the mark with the same

solvent. The mixture was stirred.

Development and Validation of a Method for Quantitative Determination 667

TABLE 2. Quantitative Determination of I Drug Substance in

Model Mixtures by GC

Kemantane

solution

concentra-

tion (taken),

mg/mL (c1)

Kemantane

found,

mg/mL (c2)

Absolute

error,

mg/mL

(d = c2 – c1)

Relative

error, %

(Y = |dx100/

c1|)

Kemantane

found, %

Metrologi-

cal charac-

teristics

(P = 95%,

n = 5)

0.422 0.428 0.006 1.42 101.42 X = 100.42

% S = 1.01

Sx = 0.45

�X = 1.25

� = 1.25 %

0.428 0.006 1.42 101.42

0.424 0.002 0.47 100.47

0.420 – 0.002 0.47 99.53

0.419 – 0.003 0.71 99.29

0.450 0.455 0.005 1.11 101.11 X = 100.27

% S = 1.31

Sx = 0.58

�X = 1.63

� = 1.63 %

0.459 0.009 2.00 102.00

0.448 – 0.002 0.44 99.56

0.444 – 0.006 1.33 98.67

0.450 0 0 100.00

0.478 0.482 0.004 0.84 100.84 X = 100.04

% S = 0.48

Sx = 0.21

�X = 0.60

� = 0.60 %

0.478 0 0 100.00

0.476 – 0.002 0.42 99.58

0.478 0 0 100.00

0.477 – 0.001 0.21 99.79

0.502 0.488 – 0.014 2.79 97.21 X = 99.00

% S = 1.16

Sx = 0.52

�X = 1.44

� = 1.46 %

0.502 0 0 100.00

0.495 – 0.007 1.39 98.61

0.502 0 0 100.00

0.498 – 0.004 0.80 99.20

0.504 0.501 -0.003 0.60 99.40 X = 99.36

% S = 0.56

Sx = 0.26

�X = 0.73

� = 0.73 %

0.501 – 0.003 0.60 99.40

0.496 – 0.008 1.59 98.41

0.504 0 0 100.00

0.502 – 0.002 0.40 99.60

0.508 0.513 0.005 0.98 100.98 X = 100.90

% S = 0.86

Sx = 0.39

�X = 1.07

� = 1.08 %

0.509 0.001 0.20 100.20

0.511 0.003 0.59 100.59

0.520 0.012 2.36 102.36

0.510 0.002 0.39 100.39

0.512 0.519 0.007 1.37 101.37 X = 100.66

% S = 0.88

Sx = 0.39

�X = 1.10

� = 1.10 %

0.510 – 0.002 0.39 99.61

0.511 – 0.001 0.20 99.80

0.518 0.006 1.17 101.17

0.519 0.007 1.37 101.37

0.524 0.529 0.005 0.95 100.95 X = 100.27

% S = 0.61

Sx = 0.27

�X = 0.76

� = 0.76 %

0.522 – 0.002 0.38 99.62

0.527 0.003 0.57 100.57

0.522 – 0.002 0.38 99.62

0.527 0.003 0.57 100.57

0.576 0.585 0.009 1.56 101.56 X = 100.69

% S = 1.18

Sx = 0.53

�X = 1.47

� = 1.48 %

0.582 0.006 1.04 101.04

0.583 0.007 1.22 101.22

0.582 0.006 1.04 101.04

0.568 – 0.008 1.39 98.61

0.600 0.605 0.005 0.83 100.83 X = 100.90

% S = 0.70

Sx = 0.31

�X = 0.86

� = 0.87 %

0.605 0.005 0.83 100.83

0.608 0.008 1.33 101.33

0.610 0.010 1.67 101.67

0.599 – 0.001 0.17 99.83

The injected sample volume was 1 �L. The test solution

was chromatographed under the conditions described above

to produce at least four chromatograms.

The content of I (X, %) was calculated using the follow-

ing formula:

XS K C

S C

x st

st x

(%) %�

� �

�

�100 ,

where Sx

is the peak area of I in the test solution (mV � min);

Sst, the peak area of the internal standard (III) in the test so-

lution (mV � min); Cst, the concentration of the internal stan-

dard (III) in the test solution (mg/mL); Cx, the concentration

of I in the test solution (mg/mL); and K, a calibration coeffi-

cient.

The calibration coefficient was calculated for model

mixtures of I and III.

The technique for preparing the model mixtures for cal-

culating the calibration coefficient was analogous to that for

preparing samples for analyzing batches of I. The formula

for calculating the calibration coefficient was

KS C

S C

st kem

kem st

�

�

�

,

where Sst

is the peak area of III (mV � min); Skem

, the peak

area of I (mV � min); Cst, the solution concentration of III

(mg/mL); and Ckem

, the solution concentration of I (mg/mL).

The validation characteristics were the accuracy and pre-

cision (repeatability and intralaboratory precision). They

were evaluated using model mixtures of I and III. Table 2

presents the analytical results.

Table 2 shows that the relative error of the result of a sin-

gle determination was less than 3% and of five repeated de-

terminations less than 1% with a relative error of the mean of

less than 1.7%. The results in Table 2 confirmed that the cho-

sen technique parameters provided the required accuracy and

precision for the developed analytical technique.

The chromatography system was optimized using the rel-

ative retention time (RRT) of I to that of the internal standard

(III) and also the number (N) of theoretical plates, the asym-

metry factor (As), and the relative standard deviation (varia-

tion coefficient) (RSD). Data for the optimization of the

chromatography system were obtained by analyzing model

mixtures of I and III with concentrations of each at

0.5 mg/mL.

Table 3 presents the results of the calculation and the ac-

ceptability criteria of these parameters.

Batches of I drug substance were analyzed using the

techniques described above for quantitative determination of

I (oximation and GC methods).

Table 4 presents results for quantitative determination of

I by oximation; Table 5, by the GC method.

Tables 4 and 5 show that the results for quantitative de-

termination of several samples of I by the oximation and GC

methods differed significantly. For example, 100.05% of I

was found in sample 1 by the oximation method; 97.10%, by

the GC method. The values obtained for sample 3 were

103.81 and 99.20%, respectively.

Apparently, such results are explained by the low speci-

ficity of the oximation method. The presence in I drug sub-

stance of technical impurity II interferes with the quantita-

tive determination. The contents of this impurity in samples

1, 2, and 3 were greater than 2.5% at 2.82, 2.74, and 2.96%,

respectively, whereas it was 1.47% in sample 4.

The GC technique did not have this drawback. In our

opinion, this was its advantage despite the higher relative er-

668 A. V. Tolkacheva et al.

TABLE 3. Chromatographic System Optimum Parameters

Model

mixture

No.

Optimum parameter

RRT As N RSD, %

1 2.15 0.02 1.50 0.09 20351 1571 0.96

2 2.15 0.01 1.58 0.02 19507 1345 0.44

3 2.15 0.01 1.55 0.17 18562 1872 0.33

Acceptability

criteria

2.15 0.02 Less

than 1.6

At least

10,000

Less

than 2%

TABLE 4. Quantitative Determination of I Drug Substance

Batches by Oximation

Parameter

Expt. No.

1 2 3 4

Result, % 99.30 100.46 102.74 100.03

100.18 100.66 104.11 102.35

100.38 100.08 104.49 99.94

99.64 100.79 103.58 98.86

100.75 100.22 104.12 99.98

Metrological

characteris-

tics,

P = 95%,

n = 5

X = 100.05

S = 0.67

Sx = 0.33

�X = 0.92

� = 0.92 %

X = 100.44

S = 0.087

Sx = 0.13

�X = 0.36

� = 0.36 %

X = 103.81

S = 0.68

Sx = 0.30

�X = 0.83

� = 0.80 %

X = 100.23

S = 1.28

Sx = 0.57

�X = 1.58

� = 1.58 %

TABLE 5. Quantitative Determination of I Drug Substance

Batches by GC

Parameter

Expt. No.

1 2 3 4

Result, % 96.62 101.75 97.46 99.48

97.01 100.20 100.07 99.42

98.81 100.24 100.72 99.66

95.94 100.60 98.53 100.08

Metrological

characteris-

tics,

P = 95%,

n = 4

X = 97.10

S = 1.23

Sx = 0.61

�X = 1.94

�X = 2.0 %

X = 100.70

S = 0.72

Sx = 0.36

�X = 1.14

�X = 1.13 %

X = 99.20

S = 1.48

Sx = 0.74

�X = 2.35

�X = 2.37 %

X = 99.66

S = 0.30

Sx = 0.15

�X = 0.48

�X = 0.48 %

ror of a determination. It allowed it to be chosen for quantita-

tive determination of kemantane.

Thus, we developed a technique for quantitative determi-

nation of I drug substance using GC. It was shown using

intralaboratory reproducibility results that the GC technique

was comparable to the previously developed oximation

method and was more specific and accurate.

The developed GC technique was validated. The tech-

nique was specific, linear, sensitive, accurate, and precise.

Optimum parameters for the chromatography system and

conditions providing precise results (technique robustness

factors) were proposed.

The developed GC technique can be used for routine

quantitative analysis of I in the drug substance in both the

pharmaceutical and chemical industries. It is planned to in-

clude it in the draft FSP for kemantane drug substance.

REFERENCES

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Klin. Farmakol., 75(6), 27 – 30 (2012).

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stracts of Papers of the IVth Convention of Russian Pharmacolo-

gists [in Russian], Kazan (2012), p. 129.

3. M. Yu. Volkova, S. V. Merinova, O. B. Stepanenko, and

B. M. Pyatin, USSR Pat. No. 1,482,392, MKI4 A61K31 / 00,

G 01 No. 31 / 00, Nov. 30, 1993, “Method of quantitative deter-

mination of 1-hydroxy-4-adamantanone and methyl-tert-butyl-

ketone” [in Russian]; Byull. Izobret., No. 43-44 (1993).

Development and Validation of a Method for Quantitative Determination 669