Volume 3, Number 13, November 2022 e-ISSN: 2797-6068 and p-ISSN: 2777-0915
Siti Saroh, Maritha
Nilam Kusuma
1UPT. PSMB-Surabaya Tobacco Institute, Surabaya., Indonesia
2Adhi Tama Institute of Technology Surabaya, Indonesia
Email:
saroh.tanwir@gmail.com, Maritha.kusuma@itats@ac.id
|
KEYWORDS Validation
method, total sugar, tobacco, phenol sulfate. |
ABSTRACT Virginia Flue Cured
Tobacco is one of the type a lot of tobacco cultivated in Indonesia, Tobacco
this many used as ingredient raw cigarette cigarettes, pipe tobacco and
tobacco Susur (chewing tobacco). Leaf tobacco
contain many compound very decisive chemistry the
quality and taste of the resulting cigarettes, one of which is is sugar. Sugar content in tobacco give mild taste in
cigarettes, but if rate too tall precisely will causes burning and irritation
esophagus. However Thus, in SNI-01-4401-2006 it is not yet formulated method
testing total sugar content. On research this conducted validation
determination total sugar content in FCV tobacco with method phenol-sulfate
by UV-Vis spectrophotometry aimed at get valid
method for determination total sugar content of Virginia Flue Cured
tobacco. Validity method showed
through the parameters of linearity, precision, determination of LOD and LOQ,
and accuracy. Test started with hydrolysis tobacco use sour 4.5% sulfate.
Solution extract be read with UV-vis spectrophotometer at a wavelength of 490
nm with dye phenol-sulphate. Linearity test results curve obtained value of r
= 0.9997 in the range work 20 – 100 mg/L, accuracy test results with 7
repetitions obtained the value of % recovery is 98.19 – 101.62%, LOD and LOQ
test results respectively of 1,009 mg/L and 3,362 mg/L, while precision test
results obtained deviation relative standard of 1.025%, result this still
under the value of 2/3 CVH is 1.86. From the results of the t-test obtained
score tcount of 0.85, the result this moresmall from table ie 2.18. Validation result method for all
parameters tested has Fulfill condition acceptance so that method phenol
sulfate could used in determining rate glucose FCV
tobacco routine |
INTRODUCTION
Tobacco is commodity agriculture that has role strategic and value
economical high in Indonesia. Tobacco plays a big role in determine development
of the cigarette industry. (Murdiyati & Basuki, 2011). Industry tobacco also
provides significant contribution for Indonesian economy, especially excise and
foreign exchange, the field work, source income and development area (Handayani, Amrullah, Tarnanda, & Rahayu, 2018).
one type a lot of tobacco cultivated in Indonesia is tobacco virginia from from the United
States. Plant it's so easy found in Lombok, West Nusa Tenggara and Jember East Java. Tobacco virginia
is also known with the name Virginia Flue Cured (FCV), because of the drying
process use Genre air heat in the oven (curing barn) (Peedin, 1999).
In the international world, tobacco virginia
many traded in form Corrosion FCV or more known as virginia
FC. Where are some big used as ingredient raw cigarettes (cigarettes) and some
small for pipe tobacco and tobacco Susur (chewing
tobacco). Meanwhile, in Indonesia, tobacco Virginia FC a lot used in form
cigarette blending white and kretek cigarettes, for shag (Murdiyati & Basuki, 2011).
Between compounds that affect quality product tobacco is sugar content
in leaf tobacco, because that in SNI-01-4401-2006 regarding Virginia Flue Cured
Tobacco, sugar content included in recommended compound for tested. Sugar
content in tobacco affect the taste of the resulting cigarette (Herwati & Rochman, n.d.). Leaf sugar content
tobacco give mild taste in cigarettes, but if rate too tall precisely will
causes burning and irritation esophagus. (Dendo et al., 2018).
However Thus, in SNI-01-4401-2006 it is not yet formulated method
testing total sugar content. Method test used During this use Luff Schrool method according to SNI-01-3996-1995 regarding DFC
tobacco that counts reducing sugar content from type monosaccharides, although
Tobacco also contains compound carbohydrate complex in form starch, cellulose,
pectin, and sugar in high numbers (Tirtosastro & Murdiyati, 2010). So that for break
compounds the Becomes monosaccharide needed long time and material chemical in
amount big, so rated not enough economical.
Because of that required development method testing rate total glucose
which has accuracy and precision high, detection limit low and cost operational
inexpensive so that testing more efficient (Nur & Apriana, 2013). On research this
conducted validation determination rate total glucose in leaves Virginia Flue
Cured tobacco with method phenol the usual sulfate (Dubois) used in the
determination of total sugar in the extract plants and food. Determination
sugar level done based on measurement absorbance solution that has been reacted
with phenol and sulfate using a UV-Visible spectrophotometer instrument at a
length of the 490 nm wave (Djajadi & Hidayati, 2017).
Because Dubois' method is n't method raw for
testing tobacco so laboratory must To do validation
method for ensure suitability show work method that. This thing in accordance
with clause 5.4.5.2 ISO/IEC 17025: 2017 which states that
"Laboratory" must validate method no standard, designed / developed
method laboratory, method standard used outside room intended scope, and
affirmation as well as modification from method raw for confirm that method
that in accordance for intended use”(Tirtosastro & Musholaeni, 2017). Because of that
validation method analysis is very important done. With use method that has
been validated so validity results measurement could accountable so that could used as base in calculation next (Sugihartini, Fudholi, Pramono, & Sismindari,
2012).
Validation method defined as evaluation to something method through
test laboratory for prove that something method testing could give valid result
with look at certain parameters (Hutagaol & Niken, 2017)
Parameters validated in research this is linearity, LOD, LOQ, and accuracy . Test results with method phenol-sulfate then
compared with results sugar content with SNI-01-3996-1995 method using
student's t. test (Indriana, 2016).
METHOD RESEARCH
Research Time and Location
Study this held from week second September 2022 to with week first
month October 2022 at the Laboratory of the Testing Technical Implementation
Unit Certification Quality Goods - Surabaya Tobacco Institute (UPT. PSMB-LT
Surabaya) which is located on Jalan Gayung Bonsari In Number 12 A Surabaya.
Tools and Materials
Equipment used in research this is a shimadzu
brand UV-Vis spectrophotometer instrument, a set of
tools reflux equipped refrigeration, equipment glass, 25 mesh sieve, scales metler brand electronics toledo,
a set of tools distillation, measuring pipette, and flask measure. Materials
used in determination rate glucose this is ingredient standard glucose (Merck),
aquadest, acid sulfate concentrated (Merck), and
phenol (Merck).
Preparation Sample
Sample Virginia Flue Cured tobacco in form dry laminate cut small and
blended until Becomes flour, then sifted with sieve size 25 mesh.
Preliminary Test
leaf sugar content test was carried out FCV tobacco with the
SNI-01-3996-1995 method was repeated 7 times. The test result data is used as
comparison test results using method phenol-sulfate through the t-test, that is
compare second mean value method is different by significant or no with see
value of t count and t table.
Hydrolysis Leaf
Tobacco
Hydrolysis sample tobacco conducted use solution sour sulfate 4.5%
(Nugroho nd) A total of 3 grams sample entered in
pumpkin reflux and added 100 ml of acid 4.5% sulfate, then heated at 100oC for
1 hour. Next solution extract filtered and diluted in a 100 ml flask. Pipette 1
mL solution extract dilute in 100 mL flask using aquadest.
Do replication testing 7 times.
Making Solution
Standard
Making solution parent
glucose 1000 mg/L
Weighed 1000 mg
standard glucose and dissolved with aquadest in
pumpkin measure 1 liter.
Making row standard
Made row standard with concentration end of 20; 40; 60; 80; 100 mg/l
with pipette solution parent 1000 mg/l 2 each; 4; 6; 8; 10 ml to in pumpkin
measure 100 ml. Then diluted with aquadest until
limit. Made blank with treatment same without addition solution standard
Validation Parameters
Linearity
Pipette 1 ml of each solution row standard, add 1 ml of 5% phenol and
shaken. After that Add 5 ml of acid sulfate concentrated with fast. Solution
silence for 10 minutes, shaken and placed in hot water bath for 15 minutes,
then be measured absorbance solution use UV - Visible spectrophotometer at long
the 490 nm wave. Note absorbance of each series and make curve calibration with
plotting score concentration (x) versus absorbance (y). From the data done
calculation for get equality regression and coefficient correlation (r).
Detection limit and
quantization limit
detection limit (LOD) and quantization limit (LOQ) tests were carried
out for determine limit Lowest concentration standard that can be analyzed by
qualitatively (LOD) and quantitative (LOQ) (Anonymous,
2019). LOD and LOQ values in
research this calculated based on standard deviation response analyte and slope
curve calibration (slope). LOD and LOQ explained in accordance equation (1) as
following:
Q = (k xS_b) /S_1, (2)
Where :
Q : LOD or LOQ
k : 3 for LOD and 10
for LOQ
Sb : deviation raw
response analytic from blank
S1 : the direction of
the linear curve Among response with concentration
S1 value is the same with score direction slope curve (slope) of
equality regression y = mx + n, i.e. m value . Whereas
deviation raw blank (Sb) value same with deviation residual standard (Sy/x), so
that formula LOD and LOQ calculations become in accordance equations (2) and
(3) as following:
LOD = (3 x S (y⁄x))/Slope, (2)
LOQ = (10 x S (y⁄x))/Slope, (3)
Precision test
Pipette 1 ml of the sample that has been hydrolyzed then 1 ml of 5%
phenol was added and shaken. Add 5 ml of acid sulfate concentrated by quickly
and let stand 10 minutes. Solution shaken and placed in a water bath filled
with warm water for 15 minutes. Then measuring absorbance solution use UV
-Visible spectrophotometer at a wavelength of 490 nm. Absorbance result note
and do calculation rate glucose in tobacco use equality regression on
determination linearity. Calculation result rate glucose with repeat 7 times,
then used for calculation ripitability that is with
calculate Horwitzt CV where range acceptance RSD
repeatability < 2/3 CVH.
Accuracy and precision
Test conducted with glucose spike method pure to in sample, that is
with add as much 0.3 gram glucose or equivalent + 80%
content glucose in sample. Then samples that have been dispike
analyzed with treatment same like sample on determination precision. Glucose
levels in the spiked sample then compared with rate glucose sample pure and
calculated acquisition return of added spikes in accordance equation (4).
RECOVERY = (A - B)/C x 100%, (4)
Where :
A : Concentration
spiked sample
B : concentration
sample
C : Concentration
spike
Criteria gain % recovery received if results analysis give acquisition
return in accordance table 1.
Table 1
Condition acceptability % recovery
|
Concentration spike in the matrix sample, % |
% Earnings return |
|
100 |
98 - 102 |
|
> 10 |
98 – 102 |
|
> 1 |
97 – 103 |
|
> 0.1 |
95 – 105 |
|
0.01 |
90 – 107 |
|
0.001 |
90 – 107 |
|
0.0001 |
80 – 110 |
|
0.00001 |
80 – 110 |
|
0.00001 |
60 – 115 |
|
0.0000001 |
40 – 120 |
Source (Harmita, 2004: 119)
Description: at least conducted repetition as much as 5 times each
example and done addition analyte as much as 50 - 150% levels analyte sample.
Significance test
Test significance two average of results testing sugar content with
method phenol-sulphate and the method of SNI-01-3936-1995 was carried out by t
test using formula in accordance equation (5) and (6) as following:
s^2=((n_1-1) s_1^2-(n_2- 1) s _2^2)/((n_1+n_2-2)), (5)
t = ((x _1-x _2))/(s√(1/n_1 +1/n_2 )),
(6)
Where :
S2 = variance combined
from two data
n1, n2 = number of data
x1, x2 = mean value
t = value tcount
degree freedom (v) = n1+n2-2 (Miller 1991).
RESULTS AND DISCUSSION
Preliminary Test
preliminary test conducted determination rate glucose leaf tobacco with
SNI-01-3996-1995 method regarding Tobacco Boyolali
Bye. Test conducted as many as 7 repetitions and obtained the appropriate data
table 2.
Table 2
Glucose Level Leaf FCV Tobacco Method
SNI-01-3936-1995
|
Test |
Glucose level ( %) |
|
1 |
11.03 |
|
2 |
11.24 |
|
3 |
11.11 |
|
4 |
10.95 |
|
5 |
11.01 |
|
6 |
10.84 |
|
7 |
10.98 |
|
Average |
11.02 |
|
SB |
0.13 |
|
% SBR |
1,141 |
|
Horwitz . CV |
2.79 |
|
2/3 CV Horwitz |
1.86 |
From the data in table 2 it can be seen rate average glucose in leaves
tested tobacco with the SNI method of 11.02. Precision testing with method this
could received because %SBR value <2/3 CVH. The
average sugar content value obtained assumed as score reference because testing
conducted with method standard for testing tobacco sugar content though no
tobacco with varieties same.
Validation Parameters Method
Linearity curve standard
Determination linearity conducted with measure absorbance every solution
in row standard. Absorbance value obtained used for make curve calibration,
that is with plotting score absorbance solution standard (y) against
concentration solution standard (x). From calculation this obtained equality
regression and coefficient correlation (r). Absorbance data results measurement
row standard glucose could seen
in table 2.
Table 2
Absorbance data standard glucose
|
Concentration (mg/L) |
Absorbance (y) |
|
20 |
0.0810 |
|
40 |
0.1686 |
|
60 |
0.2525 |
|
80 |
0.3443 |
|
100 |
0.4201 |
The data in table 2 then made chart curve calibration shown in figure 1 .
Figure 1
curve calibration solution standard glucose
Figure 1 shows equality regression linear y = 0.0043x - 0.0029, where
slope (m) = 0.0043 and intercept (n) = -0.0029 with coefficient correlation (r)
= 0.9997. Coefficient value correlation (r) obtained show score good because
enter in range acceptance score coefficient correlation (r) is r > 0.995 so
that linearity could accepted.
Calculation limit detection and limit quantization
Detection limit (LOD) is limit concentration analyte the smallest
still could detected but no could set as score true, while limit quantization (LOQ)
is limit concentration analyte the smallest you can quantized (Kan K 02, 2019).
LOD and LOQ values in
research this calculated based on standard deviation response analyte and slope
curve calibration (slopes). From calculation use equality linear
Table 3
LOD and LOQ . calculation results
|
parameter |
Results |
|
r |
0.9997 |
|
slope |
0.0043 |
|
LOD |
1.009 |
|
LOQ |
3,362 |
Precision test
Precision test aim for see proximity individual test results on testing
repeated with see scatter results measurement to the average yield measurement
on sample homogeneous
Test conducted with 7 repetitions. Result data testing precision could seen in table
Table 4
Precision test results
|
Test |
Absorbance |
Glucose level ( %) |
|
1 |
0.1461 |
11.09 |
|
2 |
0.1420 |
11.31 |
|
3 |
0.1400 |
11.11 |
|
4 |
0.1392 |
11.06 |
|
5 |
0.1396 |
11.01 |
|
6 |
0.1386 |
11.04 |
|
7 |
0.1375 |
10.95 |
|
Average |
11.08 |
|
|
SB |
0.11 |
|
|
% SBR |
1.025 |
|
|
Horwitz . CV |
2.79 |
|
|
2/3 CV Horwitz |
1.86 |
|
Based on the data in table 4, it is obtained %SBR value of 1.025 and
still under the value of 2/3 CV Horwitz is 1.86 so that precision could
accepted. There is relative test results on 7
repetitions more small from other test results possibility because error random
including fluctuations temperature heating during the hydrolysis process and
also when reading. Error random this could minimized with multiply repetition (Ariq,
Afriani, Zuliandanu, & Suhartini, 2022)
Accuracy test
Accuracy the test result (accuracy) is requirements fundamental in
testing. Test results with accuracy tall will give trust and validity results
On research this is an accuracy test conducted with standard spike
method to in sample tobacco Virginia Flue Cured that has been is known
rate the glucose through precision test, then analyzed back. Accuracy test
conducted 7 repetitions with concentration spiked standard about 90% rate
glucose sample. Added analytes in the form of standard glucose pure purpose
knowing influence treatment acid at level glucose standard. Calculation result
accuracy shown in table 3.
Table 5
Level recovery test results glucose
|
Test |
Sample rate |
Spike Rate |
Sample rate + spikes |
% recovery |
|
1 |
11.08 |
10.07 |
20.97 |
98.19 |
|
2 |
11.08 |
10.22 |
21.17 |
98.67 |
|
3 |
11.08 |
10,17 |
21.30 |
100.46 |
|
4 |
11.08 |
10.32 |
21.34 |
99.46 |
|
5 |
11.08 |
10.25 |
21.43 |
101.03 |
|
6 |
11.08 |
10,13 |
21.38 |
101.62 |
|
7 |
11.08 |
10,30 |
21.40 |
100.22 |
|
Range Recovery
(%) |
98.19 – 101.62 |
|||
From result the calculations contained in table 2 are obtained the %
returns are in the range (98.12 – 101.62). This result Fulfill condition in
accordance limit Acceptance of % recovery referred to by the laboratory of (98
– 102) % at spike concentration >10%. Difference in results acquisition
return could caused existence error systematic origin
from equipment, materials chemical, operational testing (Ariq
et al., 2022)
Error systematic no could removed by overall,
but could minimized, for example error from equipment could minimized with To
do calibration tools used and errors personnel could minimized with enhancement
competence personnel through training (Gandjar
& Rohman, 2007).
Significance Test
Instrument statistics used in calculations validation method this is the
student's t test for determine what is the average result of the sugar content
test with the SNI-01-3936-1995 method and the average results of the sugar
content test with method phenol-sulfate different by real. Hypothesis initial H
0 : 1 = 2 or no there
is difference results Among second method. The results of the t test
calculation of both data from above test method could seen
in table 6.
Table 6
Student's t test results
|
|
SNI method |
Method
phenol-sulfate |
|
|
11.02 |
11.08 |
|
|
0.13 |
0.13 |
|
|
7 |
7 |
|
|
0.1198 |
|
|
|
0.85 |
|
|
|
2.18 |
|
From result calculation obtained
score t count < t table , so
that could concluded second method give results rate glucose that is not
different by significant so that could used in
testing routine
CONCLUSION
Based on research that has been
done, can concluded that testing sugar content in leaves FCV tobacco can
conducted with method phenol-sulfate use UV-Visible spectrophotometer. Method
has Fulfill requirements validation test method for linearity, precision,
accuracy, limit parameters detection and limit quantization so that validity
results the test could accountable.
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Copyright
holders:
Ervina Anas Meliana, Naufal Febri Nugroho, Christina Dwi Astuti ( 2022 )
First
publication right:
Devotion - Journal of Research and Community Service
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