1. Page 1 of 9
RECO INDUSTRIES LIMITED
Validation report for the measurement of Dissolved solids(%w/w) in
Juices, Sauces, Spreads, Pain enzyme solutions, and Herbal
medicinesusing a Hand held Refractometer (0‐32 Brix)
Author
KIBABA IGNATIUS
Asst. Quality Manager/Technical Manager
(Supervisor & Designer of the Validation Exercise)
Analysts involved in the Validation Exercise
MOSES NDORERIRE & CHARITY BIIRA PATIENCE
(Laboratory Analysts/Technicians)
Date of start – End of the Validation Exercise
28th‐30th
/11/2016
Date of compilation of the Validation Report
1st
/12/2016

2. Page 2 of 9
Content:
1. Purpose………………………………………………………………………………………………………………………………….3
2. Principal of method………………………………………………………………………………………………………………..3
3. Validation design……………………………………………………………………………………………………………………4
3.1.Calibration….………………………………………………………………………………………………………………………….4
3.2.Sample preparation.……………………………………………………………………………………………………………….4
3.3.Determination of Recovery or accuracy….………………………………………………………………………………4
3.4.Determination of precision…………………………………………………………………………………………………….4
4. Validation results……………………………………………………………………………………………………………………5
4.1.Determination of Accuracy, Linearity, Coefficient of Determination r2
, Precision and Analysis
of Various (ANOVA)……………………………………………………………………………………………………………….5
4.1.1. Results of the determination for the accuracy/Recovery for the Refractometer……….……..5
4.1.2. Linearity of refractometer recovery for the verification salt solutions….………………………….6
4.1.3. Coefficient of determination (r2
)………………………………………………………………………………………6
4.1.4. Precision………………………………………………………………………………………………………………………….6
4.1.4.1. Intra Assay precision………………………………………………………………………………………………………..6
4.1.4.2. Inter Assay precision.……………………………………………………………………………………………………….7
4.1.4.3. Analysis of various (ANOVA)…………………………….………………………………………………………………8
5. Report summary…………………………………………………………………………………………………………………….9

3. Page 3 of 9
1. Purpose
The purpose for this study was to validate the Refractometer to measure dissolved solids (%w/w) in test
samples such as Juices, Sauces, Spreads, Pain enzyme solutions, and Herbal medicines. The validation
exercise was to help us determine the performance of the Refractometer. It included assessment of
recovery/accuracy and precision of the Refractometer to measure dissolved solids of the prepared
verification salt solutions.
Statistical tools such as Coefficient of variation (CV), Coefficient of Determination (r2
), and analysis of
various (ANOVA) were used in assessing the results obtained.
2. Basic Principles in the use of the Refractometer to measure Dissolved solids.
Fig. 1. Figure of a hand held Refractometer
When a ray of light passes from one medium to another, the speed of the light changes according to the
density of the transmitting medium. At the interface between two media, the ray changes direction as
its speed suddenly changes. This effect is known as refraction and is a familiar concept.The refractive
index (RI) of a substance is a measure of the speed of light in a substance relative to that in a vacuum
(very close to the speed in air). The RI is a physical property that depends upon temperature and the
wavelength of the light. For a particular substance the RI is a unique number when measured using a
monochromatic light source (single wavelength) at a fixed temperature. A refractometer is a device that
measures the RI of a substance, usually a liquid, but sometimes a solid.
Most modern refractometer designs utilize the concept of Critical Angle, which is attributed to Ernst
Abbe, the 19th Century physicist. The principle is illustrated below.
Incident light strikes the surface of a medium at
different angles.At low angles the light passes
through the substance and thisis called refracted
light. At a certain angle of incidence called the Critical
Angle, the light no longer passes through; it is reflected back.
From the viewing position, an areaof light and an area of dark
are therefore observed, corresponding to transmitted
(refracted) light andreflected light (no transmission),

4. Page 4 of 9
respectively. The Borderline between the two areasdefines the critical angle condition and this can be
mathematically related to the RI of the substance.
The simplest and most popular use of a refractometer is in measuring the concentration of sugar in
water. As the concentration of sugar increases the RI increases. A refractometer can therefore be used
to measure concentration of sugar provided the relationship between RI and concentration (and
temperature!) is known.
The Brix scale is more popular than RI itselfand is based on the relationship between pure sucrose in
water concentration (%weight) and RI.The Brix scale originated in the food industry where “Brix” is the
primary unit of measurement for dissolved solids.It corresponds to the % sucrose concentration in a
solution (e.g., 5° Brix=5% wt/wt. sucrose). Brix is used for testing ‘liquid food’ products even when the
food does not just contain sucrose in water, but other dissolved ingredients, the Brix scale is used as a
measure of ‘nutritional value. Thus soft drinks, juices, sauces, preserves etc. are assigned ‘a Brix value’
as part of the Quality Assurance for the product. Indeed, in the juice and soft drink industries, the Brix
value is arguably the most important parameter in quality control.
3. Design of the validation exercise.
3.1.Calibration
Before the start of analysis of each verification salt solution, the refractometer was calibrated at one
point calibration with distilled water.
3.2 Sample preparation.
Verificationsalt solutions were prepared by the supervisor of the validation exercise using General
purpose reagent 99.5% sodium chloride and distilled water at four different concentrations with in the
measuring range of the refractometer.
3.3 Determination of Recovery or accuracy
Recovery/Accuracy is defined as the nearness of a result or the mean of a set of measurements to the
true value.
In the determination of the recovery/accuracy and linearity, four different verification salt solutions of
known concentrationswere measured four times and their average values ofdissolved solids (Brix)
tabulated along with their respective expected dissolved solids (Brix)‐Table.1, pg.5.The recovery for each
verification salt was then computed and linearity of the results of analysis assessed by plotting the
expected values of dissolved solids(Brix) for the verification salt solutions against the observed values
(Fig.2.pg.6) and also computing the value of the coefficient of determination r2
.
3.4 Determination of precision.
Precision which is a general term for variability between repeated tests has got two measures:
Repeatability and reproducibility.
Repeatability precision also known as intra assay precision is the variability to be expected when a
method is performed by a single analyst on one piece of equipment over a short timescale. I.e.: Same
person, same sample, short time, same equipment and with same method.

5. Page 5 of 9
Reproducibility precision also known as Inter assay precision is the variability to be expected when a
method is performed by different analysts, using different equipment in different laboratories, over
considerable period of time.
In the determination of intra assay precision, two verification salt solutions of known dissolved solids
(Brix) were analyzed by a competent analyst in five replicates and the range of intra assay precision as a
coefficient of variation for the method determined(Tab.2.pg.6)
In the determination of inter assay precision, three verification salt solutions of known dissolved solids
(Brix) were analyzed by three analysts in three replicates. The average dissolved solids (Brix) of each
verification saltsolution by each analyst and of each verification salt solution by the three analysts was
computed (Tab.3. pg.7)
The range of inter assay precisionwhich is presented as the coefficient of variation within, and between
the three different groups/analysts wasthen determined.
An analysis of various (ANOVA) to check the level of significance of the difference between the mean of
measurements of dissolved solids(Brix) by the three different analysts was also determined (pg. 8.)
4 Validation results
4.1.Results of Accuracy, Linearity, Coefficient of Determination (r2
), Precision and Analysis of
Various(ANOVA)
4.1.1. Results of the determination for the accuracy/Recovery for the Refractometer
Table1: Measured Dissolved solids (Brix) of four verification salt solutions using the Refractometer
DETERMINATION OF THE ACCURENCY FOR THE REFRACTOMETER
Verification
salt solutions
Replicates Mean of 4
measure
ments(X)
Expected
dissolved solids
of the salt(Y)
Coefficient
of variation
%
Accuracy /
Recovery %
R1 R2 R3 R4
VSAL‐T 7.5G 3 3 3 3 3 3 0 100
VSAL‐T 17.5G 7 7 7 7 7 7 0 100
VSAL‐T 30G 11 11 11 11 11 12 0 91.66666667
VSAL‐T 50G 20 20 20 20 20 20 0 100
Average Accuracy of the Refractometer 97.91666667
The accuracy of the Refractometer to analyze for dissolved solids (brix) ranges from 91.67% to 100%. The
Average accuracy/recovery for the Refractometer was 97.92%.

6. Page 6 of 9
4.1.2. Linearity of Refractometer recovery for the verification salt solutions
Fig.2The linearity of the Refractometer to measure dissolved solids (Brix) of verification salt solutions.
Note:Each dot on the graph represents an average of four measurements.
4.1.4. Precision
4.1.4.1. Intra Assay precision.
Table2: Results of the measured brix of 2verification salt solutions measured 5 times with the Refractometer
Determination of Intra assay precision of the Refractometer to measure Brix
Verification salt
solutions
Replicates Mean of 5
measureme
nts(X)
Expected
dissolved
solids/Brix(Y)
Standard
deviation
Coefficient of
variation
%(CV)
R1 R2 R3 R4 R5
VSAL‐T 7.5G 3 3 3 3 3 3 3 0 0
VSAL‐T 50G 20 20 20 20 20 20 20 0 0
Intra Assay precision as coefficient of variation was:0 %, i.e. repeatability was 100%.
4.1.3. Coefficient of determination(r2
)
The coefficient of determination (r2
) was computed and results obtained were: 0.99536.
0
5
10
15
20
25
0 5 10 15 20 25
Expected dissolved solids (brix) of the VSAL‐T
Results of dissolved solids (brix) for the salts analysed
Linearity of the Refractometer recovery with
verification salt solutions

7. Page 7 of 9
4.1.4.2. Inter assay precision.
Table.3: Triplicate results of the brix for the verification salt solutions by three analysts using a refractometer
Determination of Inter assay precision of the Refractometer
Analysts Verification
salt solutions
Replicates Mean of 3
measure
ments (X)
Expected
Brix of the
salt(Y)
STDEV (%CV)
within
groups R1 R2 R3
1st analyst VSAL‐T 7.5G 3 3 3 3 3 0 0
VSAL‐T 17.5G 7 7 7 7 7 0 0
VSAL‐T 50G 21 21 21 21 20 0 0
2nd
analyst
VSAL‐T 7.5G 3 3 3 3 3 0 0
VSAL‐T 17.5G 7 7 7 7 7 0 0
VSAL‐T 50G 20 20 20 20 20 0 0
3rd analyst VSAL‐T 7.5G 3 3 3 3 3 0 0
VSAL‐T 17.5G 7 7 7 7 7 0 0
VSAL‐T 50G 20 20 20 20 20 0 0
Analysts Verification salt solutions
SAL‐T 7.5G:
Expect:3°
SAL‐T‐17.5G
Expect:7°
SALT50G
Expected:20°
1st
3 7 21
2nd
3 7 20
3rd
3 7 20
Mean of
measurem
ents
3 7 20.33333333
STDEV 0 0 0.577350269
(%CV)
between
group
0 0 2.84
Inter Assay precision as coefficient of variation ranges from 0 % ‐ 2.84%, i.e.reproducibility is ≥ 97.16%.

8. Page 8 of 9
4.1.4.3. ANALYSIS OF VARIOUS (ANOVA)
The means for the results of analysis of brix for the verification salt solutions by the three analysts were
subjected to the statistical test of ANOVA to check out if there was a significant difference in the results
of analysis by the three analysts.
The results of ANOVA statistical test performed at 07:34 on 29-NOV-2016
Source of Sum of d.f. Mean F
Variation Squares Squares
between 0.2222 2 0.1111 1.3477E-03
error 494.7 6 82.44
total 494.9 8
The probability of this result, assuming the null hypothesis, is 0.999
Group A: Number of items= 3
3.00 7.00 21.0
Mean = 10.3
95% confidence interval for Mean: -2.495 thru 23.16
Standard Deviation = 9.45
Hi = 21.0 Low = 3.00
Median = 7.00
Average Absolute Deviation from Median = 6.00
Group B: Number of items= 3
3.00 7.00 20.0
Mean = 10.0
95% confidence interval for Mean: -2.828 thru 22.83
Standard Deviation = 8.89
Hi = 20.0 Low = 3.00
Median = 7.00
Average Absolute Deviation from Median = 5.67
Group C: Number of items= 3
3.00 7.00 20.0
Mean = 10.0
95% confidence interval for Mean: -2.828 thru 22.83
Standard Deviation = 8.89
Hi = 20.0 Low = 3.00
Median = 7.00
Average Absolute Deviation from Median = 5.67

9. Page 9 of 9
5.0. SUMMARY OF VALIDATION RESULTS
ACCURACY: Average accuracy: 97.92%Criteria: (90‐110%),
COEFFICIENT OF DETERMINATION (r2
): Results: 0.99536, Criteria (0.995)
INTRA ASSAY PRECISION: Results: (CV): 0 %, i. e: (Repeatability is 100%), Criteria: (CV: <20%)
INTER ASSAY PRECISION:Results: (CV): 0 % ‐ 2.84% i.e. (Reproducibility is ≥97.16%).Criteria: (CV: <20%)
ANOVA:
The results of a ANOVA statistical test performed at 07:34 on 29-NOV-2016
Source of Sum of d.f. Mean F
Variation Squares Squares
between 0.2222 2 0.1111 1.3477E-03
error 494.7 6 82.44
total 494.9 8
The probability of this result, assuming the null hypothesis, is 0.999(This indicates that there is
no significant difference between the results of analysis obtained by the three analysts)
CONCLUDING REMARKS:
The use of a refractometer to analyze brix meets the criteria of method validation.
There is a high correlation coefficient between the results of analysis of brix of the verification
salt solutions and their true values.
The method therefore passes the criteria for method validation and therefore recognized as an
official method to be used in the analysis of brix in sauces, spreads, papain enzyme solutions and
herbal medicines.