IOSR Journal Of Pharmacy

(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219

Volume 10, Issue 6 Series. I (June 2020), PP. 44-56 www.iosrphr.org

Chemical Stability Profile of Two Artemether Injection

BrandsBeing Transported and Stored in an Uncontrolled
Conditions
Dr. Abdrhman Mahmoud Gamil
Associate Professor of Pharmaceutics, Al-Neelain University, Sudan
Key words: Artemether stability, Reference Thermal Exposure, Uncontrolled storage conditions, shelf life
estimation.
Received 10June 2020; Accepted 27-June 2020

ABSTRACT:The manufacturers stability data of a product may not reflect its true stability profile during its
presence in the distribution channels. WHO recommended additional study when the transportation and storage
conditions are uncertain and not complying the manufacturer labelled conditions. Artemether injection is one of
the most important antimalarial remedies in Sudan National Protocol. Samples of two brands are transported
and stored at five state capital cities and accompanied by dataloggers then returned back to the centre for testing
for its active ingredient contents at zero time, one month, 6 months and 12 months. The reference thermal
exposure was computed and the stability profile curve is plotted.it had been found that the exposure period
above 25 ͦC is 51% to 65% of the total period and up to 25% of the period is above 30 ͦC. one brand degraded
rapidly and some samples lost one of its labelled shelf life. The other brand, the artemether content increased
dramatically exceeding the pharmacopoeial upper limits. it had been found that the decrease in the peak areas of
the impurities is significantly correlated to the increase in the peak area of the active substances which may be
attributed to transformation of artemsinine or due isomerization of ά-artemether. it is recommended that the
Drug regulatory affairs should set relevant regulations regarding medicines transportation and storage
conditions for such products.

I. INTRODUCTION
In terms of efficacy, the most obvious effect is the loss of potency of a drug, which is the key factor in
determination of the shelf life of the drug product (Kommanaboyina and Rhodes, 1999). Stability of a
pharmaceutical product may be defined as the capability of particular formulation in specific container/closure
system to remain within its physical, chemical, microbiological, therapeutic, toxicological, protective and
informational specifications.If the product is not stored inaccordance with manufacturer instruction, then the
product may have expected to degrade more rapidly. Strict adherence to the storage conditions specified by the
manufacturer on the product label will help to ensure the product stability to manufacturer’s labelled expiration
date (USP-30-8/2007).A drug that passes all laboratory tests, it may be useless after few months upon entry on a
tropical country if the packaging, storage and transportation conditions have been substandard (Quick et al.,
1997).Degradation reaction is commonly oxidation, hydrolysis, dehydration, polymerization, isomerization and
photochemical reactions. This may result in lossor increase of active ingredient or formation of toxic
degradation products. Of all the many environmental factors that can be involved in drug degradation,
temperature is the most important one that cannot be controlled by package (Kommanoboyina and Rhodes –
1999).The stability of a drug product depends on the raw materials used, warehouse and transport
facilities.Increase in temperature enhances the rate constant and the effect can often be described by Arrhenius
equation.
Ea
Log K = log A  Log K versus 1/T, linear plot will be obtained. Ea usually between 50-96
2.303RT
Ks/mol.The speed of many reactions increases about two or three times with each 10 °C rise in temperature.

Estimation of shelf-life from real time data:

The Woolf’s equation has been used to estimate the shelf-life of a product from data obtained at the
same temperature conditions as those expected for the final product. The time at which drug content diverges
from its specifications is estimated by extrapolating the time course of degradation at specific temperature when
the time course of specific drug content (Yoshioka,2000). The confidence interval of the time at which the drug
diverges from specification, determined by regression analysis of n sets of time-content data is given by
Woolfsequation (Bolton, 2004):X-gx¯ ±[t(Syx)/b] [ √(1-g)/N+(X-x¯)²/∑(x-x¯)²]/1-g
44
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
Where, Syx =√ ∑y² - (∑y)²/N - b²∑(x-x¯)² / N- 2 ,g = t²( S²yx) / b²∑(x-x¯) ² ( Bolton S.and Bon,2004). The
shelf-life (the lower confidence limit of the time at the drug content diverges from the specification range) can
be estimated. When the lower limit of content specification is 90%, the shelf-life corresponds to tL at C of
C90%.

Artemether Injection:
Artemether injection is a sterile solution of artemether in suitable oil for injection. It is a clear
colourless or almost colourless oily solution. It is an antimalarial drug (IntPh, 2003). It contains not less than
95% and not more than 105% of the amount of C16H26O5 stated on the label. It is the drug of choice for the
severe malaria in Sudanese protocol (Sudanese protocol for the treatment of malaria 6/2004). Figure (1)
Artemether chemical structure:
CH3
H

O O H C16H26O5  298.4
H
CH3 O
H O CH3
O CH3
H
Dihydroartemesinin methyl ether, (3R, 5aS, 6R, 8aS, 9R, 105, 12aR)-decahydroxhlor-10-methoxy-3,6,9-
trimethyl-3,12-epoxy-12H-pyrano[6,3-;]-1,2-benzodioxypin.
“12-b-dihydroartemesinin methyl ether”.Artemether is the methyl ether of dihydroartesinin, which is derived
from artesinin the isolate substance from the extract of leafy portion of Artemisia annua (GingHao) family
asteraccae.It acts through the endoperoxides group as schizontocidal destructing the asexual erythrocytes form
of Plasmodium filciparum and P. vivax (Ch. ph1995).

Stability consideration:
The Int. Ph specifies that any of the related substances should not be more than 0.5% and not more
than one related substance should be more than 0.25% and the total of all the related substances should not be
more than 1% calculated from peak obtained from the HPLC chromatogram, disregarding any peaks less than
0.05%.
Figure 2:Dihydroartemisinin Figure 3: α-artemether and unknown related substance.
CH3 CH3
H H

O O O O
H H
H3C O H3C O
CH3 CH3
H O H O
OH
OCH3
For long-term storage at 35 °C and 30 °C RH 60% indicates a decrease in the content after 24 months
but without dropping below 90% and increase in the related substances dihydroartmesinin and other related
substances. At 40 °C and 50 °C for 12 and 6 months, an increase of unknown decomposition products is
observed, which is not correlated to pronounced decrease in drug content.At elevated temperatures a higher
decrease in content is observed. The product need to be protected from light and protected from temperature
above 30°C to avoid loss and degradation of artemether. Artemisinin and its semisynthetic artemether degrade
with activation energy 13.54 Kcal/mol (Kinghub L, 2005). The shelf-life of most products is two years.It is
recommended that, appropriate transportation and storage facility must be ensured to maintain product stability
and integrity.(WHO, 2003).

Field stability studies:

The retained samples in the manufacturer stores are not dropped off the back of a truck, they are not
left on loading dock in the blazing sun, nor were they subjected to rains or dusty weather. Thus it is unrealistic
to expect retained samples to reflect accurately the stability of the products in practice.WHO agreed that when
transportation and storage conditions did not comply the real time conditions, additional study may be needed
(WHO,2001). An inland stability testing was performed in Sudan, in 3 hospitals, where samples from IDA were
transported for Sudan in 8 months and stored for 4 months and retained back to IDA laboratories, of 15
samples, 3 showed serious loss of potency (Hogerzeilet al., 1991).Mail order distribution across USA showed

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 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
that only 8.4% of the packages experienced temperature variations with the allowable excursions
(Kommanoboyina and Rohds, 1999).

II. MATERIALS AND METHODS: MATERIALS

Product N: Artemether 80 mg/ ml ampoules - India, donated by the Scientific office. It is an oily solution in 1
ml glass ampoules. Each 8 ampoules in carton box.
Product C:Artemether 80 mg/ mlampoules - China, donated by CMS, it is an oily solution in1 ml glass
ampoule. Each 8 ampoules are contained in carton box.
HPLC grade reagents: Acetonitrile from Sotex pharmaceuticals –UK, Potasium Dihydrogen orthophosphate –
UK, Dilute acetic acid, Dibasic sodium phosphate. BDH- Germany, Phosphoric acid. BDH- Germany,
Methanol - BDH- Germany, Hexane sulphonic acid sodium salt. Sotex. U.K, Ethanol BDH- Germany, 1-
Propanol – Sotex- U.K., purified water, Eurowater.
Analytical reagents:Sulphanilic acid solution – Sotex, U.K, Aminonaphthalenesulponic acid – Sotex U.K, 0.1
Hydrochloric acid - BDH- Germany, Nitrite standard solution 20 PPm - BDH- Germany, Sodium Dihydrogen
Phosphate Monohydrate- BDH- Germany.
Reference standards:Artemether Reference Standard, (Chinese Ph. 2005).Herbstars Co. China.
Instruments:Temperature and humidity datloggers: Five devices from SATO KEIRYOKI MFG. CO. LTD.
TOKYO 101 – 0037 JAPAN SK- L 200II .No. 8171.Isocratic HPLC with UV detector-Knauer-Germany.It
consists of degasser S.N 62349. Pump S.N 60996 – 60997. Solvent organizer S.N. 62081. Dynamic Mixing
Chamber S.N. 60527. Column thermostat, jet stream 2 plus, S.N. 61884. UV detector K 2501 S.N. 60369,
software Eurochorm HPLC version 2.05. Columns:Lichro cart 250 mm x 4 lichorospher 100 RP -18 (5
micrometer) HPLC cartridge. Packed with silica gel bounded to octadecylsilyl Groups. HPLC guard Column-
Merck.
Grant Ultrasonic bath, MXB 6. S.N: CG0709004. – England.pH meter, HNNA Instruments – Portugal.
Weight Balance: AB 204 Metter Toledomeasuring range 0.01- 210grams and sensitivity 0.001 gmDryer;
Mettler LP 16 Dryer. Toledo – Switzerland, S.No. : 114490150 , with Mettler PM 600 Balance S.N.
114490149. Mixers: Fisher Scientific Mixer with Magnetic Stirrer, Scientific Industrial incorporation, N.Y-
USA. Stop watch, Micro pipette :10,20, 50, 100 nl.
Chemical Analysis Methods, Injection:
A. Principle: HLPC with U.V detector (Int P- volume 5 – 2003). B. Reagents: Acetonitrile,Hcl and ethanol
HPLC grade, Artemether reference standard.
Mobile phase: It is a mixture of 62 volumes of acetonitrile and 38 volumes of water.
C. Chromatographic conditions, Lichro cart 250 mm x 4 lichorospher 100 RP -18 (5 micrometer) cartridge,
packed with silica gel bounded to octadecylsilyl. Flow rate: 1.5 ml per minute. Detection: set the U.V detector
at a wavelength of 216 nm. Injection volume: 20 D. Prepare the following solutions:Solution A:
Dilute a volume of injection with mobile phase to obtain a concentration of 10mg/ml.Solution B: Dilute a
quantity of reference standard to obtain a concentration of 10 mg/ml of mobile phase. Solution C: Dilute a
volume of solution A with mobile phase to obtain a concentration of 0.05mg/ml.
E. Procedure: Inject 20 uL of each of solution A and B. Then inject 20 uL of solution C.
F. Calculations:Calculate the percentage of contents of artemether in the sample using peak area obtained from
solution A and B. Calculate the content of the related substances as a percentage.The area of any peak other
than the principle peak, is not greater than that obtained by solution C. and not more than one peak is greater
than half area of the principle peak solution of solution C. The sum of all areas of all peaks, other than the
principle peak, is not greater than twice the area of the main peak of solution C. Disregard any peak with an
area of less than 0.1 times the area of the principle peak in chromatogram obtained with solution C.
Validation of Methods for chemical analysis: As method ispharmacopoeialand the active ingredient is within
the required range, so partial validation by testing for linearity (Huber 2003).
Testing for linearity: Procedures described in the methods were repeated for each method using five dilutions
for each sample and three replicate injections for each dilution.
The following concentration are used, Table (5) and the linear graph is obtained.

46
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
Table (1) Validation of Methods Figure 4: linearity graph

Reference Thermal exposure: R. thermal exposure is an unofficial approach for calculating the total exposure
as a function of temperature and period. The approach was proposed by pharmaceutical manufacturing
association in USA – 1980. The time in hours for each range of temperature emphasis to compare their results
to those of the mean kinetic temperature with the potency of drugs.
Programming of Dataloggerses: Each with software and calibration certificate.They are thermistor type which
is recommended by USP. Logging data every 90 minutes intervals for 52 weeks. The readings had been barred
by adjusting the screen not to display the recorded data.
Storage at the sites: The products were stored in the normal storage conditions at the hospital pharmacies
among the stored medicines intended to be dispensed to patients without altering the existing conditions. The
datalogger were placed side by side with the samples on the shelves. Five stations were selected which are
Centre and four different cities code as station 1,2,3,4 and 5.
Frequencies of testing: Initial testing, after a month, after six months and after 12 months.

III. RESULTS
Artemether 80mg/ml Injection – Indian brand N:
Appearance: It is a clear, colourless, oily solution in a colourless glass ampoule, with a break line and loosely
adhesive label.
Extractable volume: One ml ampoule.
Chemical assay for Artemether contents:
The following table represents the content percentage of Artemether in product N when stored at different
stations in their usual conditions throughout 52 weeks. Tests were done in triplicates with maximum SD 2.

Table (2) Contents of Artemether in product N at different stations during 52 weeks

Time
Station 1 Station 2 Station 3 Station 4 Station 5
0
103.63% 103.63% 103.63% 103.63% 103.63%
Transport
98.48% 103.05% 100.87% 102.03% 100.86%
6 months
82.14% 91.23% 88.24% 94.82% 86.41%
12 month
81.33% 91.8% 84.21% 88.48% 82.59%

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 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
Figure 5: Artemether in Product N HPLC peak

Artemether 80mg/ml Injection – Chinese brand (Product C):

Tests were performed for artemether 80mg/ml I.M injection– China.It is a clear, colourless, oily solution in a
colourless glass, printed ampoule, with a break line at the neck of the ampoule.
Extractable volume: 1.2 ml.
Chemical assay for Artemether contents:
Table (3) Contents of Artemether in product C at different stations during 52 weeks
Time Parameter Reference Station Station Station Station Station
Month standard 1 2 3 4 5
00 Mean content 100 % 100.67% 100.67% 100.67% 100.67 100.67
STD ----------- 0.25 0.25 0.25 0.25 0.25
1 Mean area 9.40783 9.83277 9.51785 9.61611 9.46356 9.79266
Content% 100% 104.5169 101.1695 102.2139 100.5924 104.0905
Mean area 9.85038 10.2802 10.5952 10.0601 10.4086 9.94664
6 Content% 100% 104.51% 105.95% 102.27% 105.81% 101.12%
STD 0.019714 0.019714 0.019714 0.019714 0.019714 0.019714
Mean area 10.3268 11.24468 11.01138 10.80808 9.831813 11.24463
12
STD 0.050688 0.493769 0.167257 0.04336 0.432003 0.610713
Content% 100% 108.8883 106.6291 104.6604 95.20677 108.8878

Table (4): Area of Secondary Peaks in the assay of artemether in product C chromatogram

Time Station 1 Station 2 Station 3 Station 4 Station 5

00 Retentim Retentim Retentim Reten Reten Reten Reten Reten Reten Reten
e 0.55 e 0.80 e 0.55 time time time time time time time
0.80 0.55 0.80 0.55 0.80 0.55 0.80
3.767 5.734 3.913 6.581 10.561 26.96 4.39 8.491 10.196 26.982
6 5.588 13.997 5.287 11.739 6.978 12.119 6.766 12.51 5.586 8.498
month 7
s
12 1.72275 1.0175 4.529 negligi negligi 1.7002 3.311 0.864 4.1957 1.7227
month bl bl 5 5 6 5 5
s

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 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..

Figure 8: Product C initially Figure 9: Product C finally

IV. DISCUSSION
Transportation:
Table (5) shows the MKT, vehicle and change in potency for the five stations
station Vehicle MKT ͦC Time Product N Product C
hours
1 Small car 48.3 3 _ 5.2% + 3.8%
2 Indirect distribution truck 35.9 156 _ 0.7% + 0.4%
Passengers buss 45.2 21 --------- -----------
3 Truck with other 33.1 42 _ 2.9% + 1.5%
4 Airplane 30.6 72 _ 1.7% ×
5 Passengers buss 34 25.5 _2.9% + 4.2%
Artemetherinjection was greatly affected by transportationresulting in average loss of potency of 3.13% for
product N. Product C, the average increase in potency was 2.54%.

Thermal Exposure Periods:

Table (6) Thermal Exposure Periods and change in potency for the Five Stations
Station Total Exposure Period Over Period Over 30°C Product N Product C
Hours 25°C % %
1 7773.5 hours 51% 10.2% ¬ 8.2% + 22.3%
2 8700 hours 57% 3% ¬ 5.9% +11.8%
3 9003 hours 65% 0.4% ¬ 4% +19.4%
4 7927.5 hours 60.9% 25.2% ¬ 5.5% + 15.1%
5 5869.5 hours 55% 19.5% ¬ 8.2% +21%

From the above table, it could be concluded that all samples were expoesd at all stations to temperature more
than 25°C % for the most period of storage with a considerable excursions over 30°C % and this is

49
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
contradicting to the labeled storage instructions for artemether.The FDA specifies two excursions up to 30°C %
after which the storage facility will be disqualified.
Artemether 80mg/ml Injection- Indian brand (Product N):
The product retained its appearance, clarity colour and the extractable volume but the adhesive label was getting
loosely attached to the glass ampoules. Some labels were completely detached.

Chemical Stability:
The International Pharmacopoeia specifies the limits for Atemether injection 95%- 105%.From table (3) it is
clear that the potency of the product came below the specified limit within the first six months of storage at all
stations.
6 months
82.14% 91.23% 88.24% 94.82% 86.41%
12 month
81.33% 91.8% 84.21% 88.48% 82.59%
Stability Profile of Product stored at station 1:
The worst results obtained for the product were those samples stored at station 1 where the concentration of the
active ingredient reached 81.33% in 12 months.

Table (7) Analysis of Results of Product stored at station 1

Product Content in mg
1K Content % Ln content Y² X² ( X- x)²
0.00 103.7 82.96000000 4.41835856 19.52189240 0.00 37.3321
0.00 103.6 82.88000000 4.41739378 19.51336779 0.00 37.3321
0.00 103.6 82.88000000 4.41739378 19.51336779 0.00 37.3321
1.00 98.48254 78.78603200 4.36673572 19.06838087 1.00 26.1121
6.00 82.14486 65.71588800 4.18534072 17.51707697 36.00 0.0121
12.00 84.11356858 67.29085486 4.20902434 17.71588591 144.00 34.6921
12.00 81.68329008 65.34663206 4.17970590 17.46994143 144.00 34.6921
12.00 75.63582136 60.50865709 4.10278645 16.83285663 144.00 34.6921
12.00 83.88803889 67.11043111 4.20633949 17.69329189 144.00 34.6921
55.00 697.92321525 38.50307874 164.84606167 613.00 276.8889
On plotting Ln content against time using LSR analysis graph (10).

Graph (10) Stability Profile of Product N stored at station 1

Artemether Ln Content in 1K = 4.3983-0.0197*x
4.45
Artemether Ln Content in 1K

4.40

4.35

4.30

4.25

4.20

4.15

4.10

4.05
-2 0 2 4 6 8 10 12 14
time:Artemether Ln Content in 1K: y = 4.39826215 - 0.0196596481*x
time

The LSR line equation was: Ln C = 4.9826215 – 0.0196596481 × t

Thus, t95= 3.43 months.
The ICH, FDA and EMRO stated that 5% one- sided lower CI was appropriate enough for drugs known to
decrease with time. By application ofWoolfs equation the 5% lower one-sided confidence interval for Ln 76 mg
intercepts the time axis at 2.3 months. Thus, the product will lose 9 months from its labelled shelf life which

50
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
will be only 15 months. This may be due to transportation and storage conditions that were below the labeled
storage directions.

Determination of Reaction order and Rate constant of Artemether Degradation Reaction in Product N:
Using the substitution method, results were substituted in the integral equation of zero, first and second order of
reaction to get the rate constant values
Zero order first order second order
C 0  Ct 2.303 C 1 x
K0  K1  log 0 K 2  .
t t Ct at (a  x )
The substitution is repeated for each equation at each time interval, the order at which K remains constant is the
related order. Table 13 shows the calculations of the rate constant K.

Table (8) Analysis of Rate Constant Values:

Product N C (a) Ct(a-x) C-Ct(x) log(c/ct) K° K¹ K¹¹
103.6333 82.90667
1 103.0546 82.44367 0.456333 0.002397 0.456333 0.005521 0.00006677
6 91.22955 72.98364 9.91636 0.055329 0.009222 0.021237 0.00027316
12 91.806 73.4448 9.455196 0.052593 0.004383 0.010094 0.00012941
1 98.32629 78.66103 4.23897 0.022795 4.23897 0.052497 0.00065005
6 86.88469 69.50775 13.39225 0.076521 0.012754 0.029371 0.00038736
12 82.28171 65.82537 17.07463 0.100161 0.008347 0.019223 0.00026075
1 100.8566 80.6853 2.214699 0.01176 2.214699 0.027084 0.00033110
6 86.41297 69.13038 13.76962 0.078886 0.013148 0.030279 0.00040045
12 82.58555 66.06844 16.83156 0.09856 0.008213 0.018915 0.00025609
1 98.48254 78.78603 4.113968 0.022105 4.113968 0.050909 0.00062988
6 82.14486 65.71589 17.18411 0.100884 0.016814 0.038723 0.00052572
12 81.33018 65.06414 17.83586 0.105213 0.008768 0.020192 0.00027556
1 100.8663 80.69304 2.20696 0.011718 2.20696 0.026988 0.00032992
6 88.24473 70.59578 12.30422 0.069776 0.011629 0.026782 0.00035040
12 84.21067 67.36854 15.53146 0.090097 0.007508 0.017291 0.00023175
1 102.0308 81.6246 1.275396 0.006733 1.275396 0.015507 0.00018848
6 94.82384 75.85907 7.040929 0.038547 0.006425 0.014796 0.00018660
12 88.47707 70.78166 12.11834 0.068634 0.005719 0.013172 0.00017210
mean 0.812181 0.024366 0.000313642
St.d 1.427292 0.012764 0.000160087
CV 175.7357 52.38629 51.0412156

For the zero order:There was a significant difference in values of K0 sig value 0.00 between the groups and within
the groups.
For the first and second order:
There were insignificant differences in K1 and K2 values between groups and within the groups, so the reaction
behaved as second order as well as first order. Thus, it could be assumed to be pseudo-first order with K1 value
2.4 × 10¯²mg.month¯¹.
Stability profile of Artemether Injection Chinese brand ProductC
The product retained its physical characters. The extractable volume was 1.2 ml meaning that there was 0.2 ml
overage constituting 20%, so, if the concentration of artemether initially was 100%, the content of the ampoule
will be 120% which above the International Pharmacopoeia limits.
Artemethercontent:
As presented in table 4, the concentrations of Artemether increased above 105% in product 2 at all stations after
six months of storageand continued to increase at for stations and reached 108.88% at station 1, 2 and 5.
Stability Profile of Product C being stored at Station 2:
To analyze the results of potency against time and to estimate the expiration date.

51
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
Table (9) Analysis of Results of Product C at Station 2
Product 2 B Content % Content Y² X² ( X- x)²
0.00 100.4 80.32000000 6451.30240000 0.00 37.33210000
0.00 100.9 80.72000000 6515.71840000 0.00 37.33210000
0.00 100.7 80.56000000 6489.91360000 0.00 37.33210000
1.00 101.1695 80.93560000 6550.57134736 1.00 26.11210000
6.00 105.9586 84.76688000 7185.42394493 36.00 0.01210000
12.00 105.8227 84.65816000 7167.00405459 144.00 34.69210000
12.00 106.0677 84.85416000 7200.22846931 144.00 34.69210000
12.00 105.5855 84.46840000 7134.91059856 144.00 34.69210000
12.00 109.0406 87.23248000 7609.50556695 144.00 34.69210000
55.00 748.51568000 62304.57838170 613.00 276.88890000

The LSRL obtained (graph 11 ) was used to estimate the expiration date.

Graph (11) Stability Profile of Artemether in product C / Station 2

Stability Profile of Artemether in product 2 B
Artemether Content in 2 B = 80.7443+0.3967*x
88

86
Artemether Content in 2 B

84

82

80

78

76
-2 0 2 4 6 8 10 12 14
time:Artemether Content in 2 B: y = 80.7443304 + 0.396667384*x
time

The LSRL equation was C = 80.7443304 + 0.396667384 × t

105% concentration equals 84 mg, thus the time required for concentration to reach 84 mg was:t(105) = 8.207
months.
As the potency may decrease or increase, the 5% two-sided confidence intervals were considered to
demonstrate the product shelf life.
The 5 % two-sided confidence interval for X at a given Y could be given by Woolfs inverse prediction equation.
X-gx¯ ±[t(Syx)/b] [ √(1-g)/N+(X-x¯)²/∑(x-x¯)²]
1-g
Where, Syx =√ ∑y² - (∑y)²/N - b²∑(x-x¯)²
N- 2
g = t²( S²yx) / b²∑(x-x¯) ²
and t value of 5% two- sided CI at d.f 7 is 2.36
The 84 mg content line intercepts the upper band of the two-sided confidence interval at 6.1 months .One-sided
CI at t-value 1.89 for the d.f. 7, is 6.5 months.
Thus, the conformance period of this product at Station 2 will be 6.5 months and thus lose 3 months from its
labelled shelf life due to improper transportation and storage and the quality of the product.

Stability Profile of Product C being stored at station 5:

The LSRL equation was C= 80.6925035 + 0.508739422 × t.
Thus, t(105)= 6.5 months.

52
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
The upper confidence interval of results at time zero exceeded the International Pharmacopoeia limits. By
application of calculations of the inverse prediction, the upper band of the 5% one-sided confidence interval
intercepts the line of 84 mg at 0.6 months. This finding was in conformance with the results of the extractable
volume, that the addition of 0.2 ml overage constituted 20% of the required potency.

Stability Profile of Product C being stored at Station 1:

The LSRL equation was: C= 81.0761335 + 0.5014541 × t, Thus, t(105) = 5.8 months.
The upper confidence interval of results at time zero exceeded the International Pharmacopoeia limits. By
application of calculations of the ICH and EMRO, the upper band of the 5% two sided confidence interval
intercepts the line of 84 mg one month before commencing of the study and the one-sided CI at 1.5 months.
This finding was in conformance with the findings obtained from the same product being stored at station 5 and
those of the extractable volume, that the addition of 0.2 ml overage constituted 20% of the required potency.

Stability Profile of Product C being stored at Station 3:

To analyze the results of potency against time and to estimate the expiration date, table (17) was prepared.

Table (10) Analysis of results of product C stored at Station 3

Product 2
Th Content % Content Y² X² ( X- x)²
0.00 100.4 80.32000000 6451.30240000 0.00 37.33210000
0.00 100.9 80.72000000 6515.71840000 0.00 37.33210000
0.00 100.7 80.56000000 6489.91360000 0.00 37.33210000
1.00 102.2139 81.77112000 6686.51606605 1.00 26.11210000
6.00 102.2738 81.81904000 6694.35530652 36.00 0.01210000
12.00 104.7188 83.77504000 7018.25732700 144.00 34.69210000
12.00 104.2424 83.39392000 6954.54589297 144.00 34.69210000
12.00 104.4622 83.56976000 6983.90478646 144.00 34.69210000
12.00 105.2185 84.17480000 7085.39695504 144.00 34.69210000

The LSRL obtained (graph 13) was used to estimate the expiration date.

Graph (12) Stability Profile of Artemetherin Product C stored at Station 3

Artemether Content mg in 2 Th = 80.7155+0.2484*x
90

88
Artemether Content mg in 2 Th

86

84

82

80

78
-2 0 2 4 6 8 10 12 14 16 18
time month:Artemether Content mg in 2 Th: y = 80.7154646 + 0.248445425*x
time month
The LSRL equation: C = 80.7154646 + 0.248445425 × t
Thus t(105) = 13.2 months.

53
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
The 84 mg content line intercepts the upper band of the two-sided confidence interval at 12 months. From
Woolfs equation at t value 2.36 the upper band of the two-sided confidence interval at 11.3 months and the
One-sided CI at 11.6 months.
Thus, the conformance period of this product at Station 3 will be 12 months, this gives a loss of 8months from
the labeled shelf life.

Comment on the Chemical Stability Profile of Product C:

There was no significant difference between the graphical and calculated intercept for all stations and within the
stations.Using Statistica software, one sample t test for the intercepts obtained from the LSRL equations and the
measured values at zero time, yields the following results.
Reference value d.f mean St.d. St.error CI - 95% CI+95% t- value P

80.6 5 81.037 0.4046 0.16518 80.6 81.5 2.64 0.05

Using Statisticasoft ware , t-sample test tool to compare the slopes of LSRL with zero
Ref.value d.f mean St.d St.error CI-95% CI+95% t-value P
00 5 0.4431 0.133 0.054194 0.303 0.582 3.17 0.0004
The slopes of the LSRL of different stations were significantly different from zero. All the slopes are positive
except that of station 4 which was negative. Artemether contents increased with time for five stations. The areas
under the main peakand secondary peaks in the chromatogramsas in table (9) are correlated using Stastica
Software, Matrix correlation tool, the area of the main peak, secondary peak 1 and secondary peak 2 were
correlated, resulting in table (118).

Table (11) Correlation Analysis

Station main Peak and secondary 1 main Peak and secondary 2
1 - 0.85 - 0.84
2 - 0.73 - 0.49
3 - 0.81 - 0.82
4 - 0.13 - 0.34
5 - 0.65 - 0.93

From the above table there was a marked significant negative correlation between the Artemether peak
area and the other two secondary peaks. As the secondary peaks decrease, the main peak increases. Knowing that
the related substances supposed to be present were α-artemether and dihydroartemisine, it is likely to suppose that
transformation took place between the two isomers, α-artemether and β-artemether where the α-isomers was known
to be less effective. Also dihydroartemisine was the intermediate substance in synthesizing artemether. Artemether
is the methyl ether of dihydroartemisine, so it likely to suggest that dihydroartemisine was transformed to
artemether during the inappropriate conditions of transportation and storage. These related substances were
supposed to increase with time but it had been found to decrease with time.
Samples being stored at station 4 showed a negative slope despite of the negative correlation between
peaks, but it is obvious that after 6 months of storage the drug content was above 84 mg constituting the peak then
the results of the last point were responsible for the negative slope. This could be attributed to the severe
transportation conditions during transportation from station 4 to station 1, where analysis took place, in summer.
The excessive overage had been added to compensate the extension of the shelf life to three years but it
did not regard the upper Pharmacopoeial limits.

Determination of rate constant of degration of Artemether in Product 2:

Artemether in ProductC increased with time, marked significant negative correlation was found to take
place between the main and secondary peaks, indicating the conversion of α-artemether and dihydroartemesinine ,
the related substances, into artemether by time at tropical conditions.

Table (12)Determination of rate constant of degration of Artemether in Product C:

Time/
month Percentage C (a) Ct(a-x) C-Ct(x) log(C/Ct) K0 K1 K2
0 100.6667 80.53333
1 101.1695 80.93556 -0.40556 -0.00218 -0.40556 -0.00502442 -0.00006222
6 105.9586 84.76685 -4.23685 -0.02227 -0.70614 -0.00854733 -0.00010344
12 106.6291 85.30329 -4.77329 -0.02501 -0.39777 -0.00479947 -0.00005790
1 104.0905 83.27242 -2.74242 -0.01454 -2.74242 -0.03349369 -0.00040896
6 101.1203 80.89626 -0.36626 -0.00197 -0.06104 -0.00075644 -0.00000937
12 108.8878 87.11024 -6.58024 -0.03411 -0.54835 -0.00654657 -0.00007817
54
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
1 104.5169 83.6135 -3.0835 -0.01632 -3.0835 -0.03758198 -0.00045794
6 104.5114 83.60911 -3.07911 -0.0163 -0.51319 -0.00625491 -0.00007622
12 108.8883 87.11062 -6.58062 -0.03411 -0.54839 -0.00654694 -0.00007817
1 102.2139 81.77112 -1.24112 -0.00664 -1.24112 -0.01529710 -0.00018848
6 102.2738 81.81903 -1.28903 -0.0069 -0.21484 -0.00264716 -0.00003261
12 104.6604 83.72836 -3.19836 -0.01691 -0.26653 -0.00324625 -0.00003953
1 100.5924 80.4739 0.056097 0.000303 0.056097 0.00069696 0.00000866
6 105.8167 84.65339 -4.12339 -0.02169 -0.68723 -0.00832406 -0.00010081
12 95.20677 76.16541 4.364586 0.0242 0.363715 0.00464436 0.00005930
mean -0.83045 -0.01009654 -0.000122731
St.d 1.018891 0.01245462 0.000152215
CV -122.691 -123.355259 -124.0229152

The values of K2 were insignificantly different with sign. value 0.312.

Values of K1 were insignificantly different with sign. value 0.309.
K0 values were insignificantly different with sign. value 0.305.
The reaction order was indefinite that the reaction might be second, first or zero order, the difference in CV was
only 0.7%. All orders may take place because there might be conversion of α and β- artemether and
dihydroartemesinine to atemether and the degradation of artemether.

V. CONCLUSION
Artemether 80 mg/1 ml injection – INDIA (Product N):
This product exhibited a marked instability profile, although the manufacturer instructed storage
conditions below 25°C, the product was exposed to temperature over 25 for 51 – 61% of the storage period and
to the drastic conditions of transportation. The samples being collected from all stations were of potency less
than 95%, the specified limits of the international Pharmacopoeia. Thus, the product failed stability testing from
the first six months. The reaction follows pseudo- first order reaction of kinetics of rate constant K1 = 2.4 ×
10¯² mg. month¯¹.
The worst results obtained from samples at station 1 where the LSRL first order equation was Ln C =
4.9826215 – 0.0196596481 × t, , t(95) = 3.43 months. On applying ICH guidelines,t(95) = 2.3 months, the shelf
life will be lessened nine months.This might be attributed to artemether inherited properties and quality, lack of
adequate overage, the non-complying transportation and storage conditions with those specified by the
manufacturer and the narrow range specified by the Int. Ph.

Artemether 80 mg/1ml injection – CHINA (ProductC):

The product contains excessive overage 0.2 ml; 20% of the labeled volume indicating improper filling
and poor QC & QA measures. It is out of the int.Ph range. Only after six months the artemether contents of the
samples at station 2 were above the pharmacopoeial limits. The reaction order was indefinite that the reaction
might be second, first or zero order, the difference in CV was only 0.7%. All orders of reaction may take place
because there might be conversion of α and β- artemether and dihydroartemesinine to atemether and the
degradation of artemether. Samples returned from station 2 yielded the zero order equation: C = 80.74
+0.39667384 × t, t(105) = 8.2 months and in accordance to ICH guigelinest(105) was 6.5 months and the shelf
life is 4 months out of 36 months.
Samples at station 1 yielded the zero order equation: C = 81.07 + 0.5014541 × t, t(105) = 5.8 months
and in accordance to ICH guidelines t(105) was 1.5 months and the shelf life will be18 months less out of 36
months. Samples returned from station 3 yielded the zero order equation: C = 80.71 + 0.248445 × t, t(105) =
13.2 months and in accordance to ICH guigelinest(105) was 11.6 months with a loss in shelf life of 8 months
out of 36 months. Samples returned from station 4 yielded the zero order equation:C = 81.31 – 0.37214532 × t,
t(95) = 14.3 months and in accordance to ICH guigelinest(95) was 8.5 months for one-sided CI and 7.5 months
for the two-sided CI with a loss in shelf life of 12 months out of 36 months. Samples returned from station 5
yielded the zero order equation: C = 81.07 + 0.508739 × t, t(105) = 6.5 months and in accordance to ICH
guigelinest(105) was 0.6 months with a loss in shelf life of 19 months out of 36 months.
There was a markedly significant negative correlation between the two secondary peaks of α-
artemether and dihydroartemesinine and the main peak of artemether.in samples from all stations. It was
suggested that during storage α-artemether and dihydroartemesinine undergo degradation to aremether resulting
in increased potency. The manufacturer added excessive overage to achieve 36 months shelf life disregarding
the global recommendation of Int.Ph and ICH guidelines.The product contains excessive amount of related
substances.

55
 Chemical Stability Profile of Two Artemether Injection BrandsBeing Transported and Stored ..
Recommendations:
The DRA should revise the shelf lives of the products (two similar products having two different shelf lives)
and should set regulations for the distributors and pharmacies and enforce them to adhere. DRA should conduct
a regular post-marketing surveillances.

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