Syafrinal., et al.

Chempublish Journal, 9(2) 2025, 313-329

Article

Verification of The Method for Determining Calcium Content in

Animal Feed

Syafrinal^1*, Hafnimardiyanti¹, Selfa Dewati Samah¹, Pevi Riani¹, Renny Futeri¹

Nurmaliza¹

,

¹Chemical Analysis, ATI Padang Polytechnic Jl. Bungo Pasang Tabing, Padang, Sumatera Barat,

25171, Indonesia

Abstract

Calcium is one of the essential macromineral elements in animal feed. To ensure that animal feed meets

nutritional and safety standards, accurate and validated analytical methods are needed to determine

calcium levels, specifically using the ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy)

technique, which is known for its high sensitivity, enabling the rapid and precise detection of calcium

elements at low concentrations. This study aims to verify the method for analyzing calcium content in

animal feed, referring to method verification parameters, including linearity, accuracy, precision

(repeatability test), limit of detection (LoD), method detection limit (MDL), and limit of Quantitation (LOQ).

The verification process was conducted using calcium standard solutions at various concentrations and

analysis of animal feed samples according to SNI 3148.2:2009 using Inductively Coupled Plasma (ICP-

OES) with a linearity test parameter coefficient of correlation (r) value of 0.9991, precision with a % RSD

value of 2.18%, accuracy (recovery) reaches 90-99%, with a detection limit (LoD) of 0.0745 mg/L and a

Limit of Quantitation (LoQ) of 0.9060 mg/L, all of which meet the acceptance criteria set by AOAC. These

results prove that the ICP-OES technique is suitable for use as a test method for determining the calcium

content in animal feed, can be adopted as a standardized routine procedure.

Keywords: Animal Feed, calcium, ICP-OES, method verification

*

Corresponding author

Email address: rinal1450@gmail.com

DOI: https://doi.org/10.22437/chp.v9i2.48076

Received August 29^th2025; Accepted December 01^st2025; Available online December 25^th2025

(https://creativecommons.org/licenses/by/4.0 )

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Graphical Abstract

Introduction

reproducible [5]. One of the analytical

methods currently relied upon is Inductively

Animal feed is one of the most important

components in modern livestock production

Coupled

Plasma

Optical

Emission

Spectroscopy (ICP-OES). This technology

allows for the simultaneous detection of

minerals with high sensitivity and low

detection limits, making it highly suitable for

complex matrices such as animal feed [6].

However, the use of the ICP-OES method for

determining mineral elements must first

systems

[1].

Global

animal

protein

consumption increased by approximately

14% over the past two decades. This

underscores the need for feed quality

assurance,

including

essential

nutrient

content such as calcium (Ca) [2]. Calcium is

one of the essential macromineral elements

in animal feed, playing a primary role in bone

undergo

a

verification stage to meet

analytical

validity standards. Method

formation,

transmission, and blood coagulation [3].

Calcium requirements vary greatly

muscle

contraction,

nerve

verification is an important process in

ensuring that the method used meets

performance

accuracy, precision,

specifications

linearity,

such

limit

as

of

depending on the livestock species, age, and

physiological stage. Therefore, accurate

determination of calcium levels is crucial in

feed formulation [4].

detection, and limit of quantitation [7].

International regulatory bodies such as

AOAC and SNI have emphasized the

importance of analytical method verification

processes in ensuring the quality of

laboratory test result.

In practice, the analysis of mineral content

such as calcium in feed materials requires

methods that are not only sensitive and

accurate, but also precise, selective, and

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Various previous studies have developed

methods to measure calcium in various food

materials using ICP-OES, such as in milk [8],

nuts [9], persian tahini (ardeh) ([10] and

sorghum [11] but there are no publications

that can be detected and measured with a

certain degree of confidence and the

method detection limit (MDL) to determine

the actual detection limit of the entire

analytical method [16].

that

comprehensively

examine

the

This research also contributes scientifically

by presenting empirical data that fills a gap

in the literature regarding the application

and validation of ICP-OES in livestock feed

matrices. Additionally, the results of this

study will serve as a reference for testing

laboratories, certification bodies, and feed

manufacturers in establishing accountable

verification of this method's performance

parameters in the context of animal feed.

Some other studies are still limited to

determining the general levels of mineral

elements

verification aspects of the methods used.

This research gap underscores the

importance of conducting a study to verify

the ICP-OES method for determining Ca

levels in animal feed. Therefore, verifying

this method is of significant value both

scientifically and practically, especially in

supporting the livestock feed industry, which

relies on accurate and reliable analytical data

[12].

without

emphasizing

the

mineral

testing

protocols.

With

this

background, the main objective of this

research is to verify the method for

determining the calcium (Ca) content in

animal feed using ICP-OES based on the

parameters of linearity, limit of linearity,

precision, accuracy, limit of detection,

method

Quantitation,

detection

as

limit,

regulated

and limit

in

of

SNI

This research not only determines the Ca

content using the ICP-OES technique but

also conducts a thorough verification of the

3148.2:2009 and to confirm that the ICP-OES

technique meets AOAC criteria for calcium

determination in animal feed. The results of

this research are expected to provide

theoretical benefits in the development of

analytical science, as well as practical

benefits for testing laboratories and the feed

industry in ensuring the quality of their

products.

method's

performance

parameters

according to SNI 3148.2:2009 guidelines,

which have not been systematically studied

in previous research. This standard also

includes verification parameters such as

linearity,

detection

accuracy,

limit

precision,

and

method

limit of

(LOD),

Quantitation (LOQ), which serve as a

reference for validating laboratory test

results. Linearity parameter verification is

performed to ensure that the instrument's

emission signal is directly proportional to the

calcium concentration within a specific

range, while the limit of the linearity curve is

needed to determine the maximum limit of

the calibration curve [13]. Precision indicates

the reproducibility of results under the same

testing conditions [14], while accuracy

describes the closeness between the test

result values and the true value [15].

Meanwhile, LOD and LOQ are important for

knowing the minimum concentration limit

Materials and Methods

Materials

The equipment used in this experiment

consists of two types: main equipment and

supplementary

equipment.

The

main

equipment used is the ICP-OES instrument

(PerkinElmer Optima 8300) with a plasma

power of 1400 W. Plasma gas flow 12 L/min,

Auxiliary gas flow 1 L/min, Nebulizer gas flow

0.8 L/min and axial view observation mode.

Calibration was internal using a single-

element calibration curve. Meanwhile, the

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supplementary equipment includes the

Mettler Toledo analytical balance, furnace,

100 mL and 200 mL volumetric flasks,

micropipette, 10 mL measuring pipette,

porcelain crucible, funnel, stirring rod,

spatula, compressor, 0.45 μm filter paper, 50

mL beaker, rubber bulb, and spray bottle.

The materials used in the experiment consist

of test materials and chemicals. The test

materials include livestock feed samples and

dissolve

the

remaining

minerals.

The

solution was then cooled and filtered using

0.45 μm filter paper. The obtained filtrate is

placed into a 100 mL volumetric flask and

diluted with distilled water to the mark.

Preparation of Precision and Accuracy Test

Solution

The prepared test sample of 95 mL was

placed into a 100 mL volumetric flask and 5

mL of a 10 mg/L standard solution was

added. For precision and accuracy testing,

the test solution was prepared in ten

replicates.

a

standard

calcium

solution

with

a

concentration of 1000 mg/L. The chemicals

used include concentrated nitric acid (HNO₃),

HCl solution with concentrations of 0.5 M

and 3 M, demineralized water (distilled

water), and argon gas.

Preparation of Limit of Detection Test

Solution

Preparation of 100 mg/L Calcium Standard

Solution

The instrument's detection limit test is

conducted using a solvent blank. The

preparation of the blank involves using 5 mL

of concentrated HNO₃, which is added to a

100 mL flask that has previously been

supplemented with a small amount of

distilled water, then brought to the mark

with distilled water and homogenized. The

Limit of Detection test solution was made in

ten replicates.

A total of 10 mL of standard calcium solution

with a concentration of 1000 mg/L was taken

using a pipette, then placed into a 100 mL

volumetric flask. Next, the volume of the

solution was adjusted with distilled water

until it reached the mark, then shaken until

homogeneous.

Preparation of Calcium Standard Series

Solutions (0; 2; 4; 8; 16; 20) mg/L

Preparation of Method Detection Limit and

Limit of Quantitation Solutions

5 mL of concentrated nitric acid (HNO₃) was

pipetted into each 100 mL volumetric flask

that contained a small amount of distilled

water. After that, a standard calcium solution

with a concentration of 100 mg/L was added

to each volumetric flask in amounts of 0, 2,

4, 8, 16, and 20 mL, respectively.

A 20 mg/L standard solution of 5 mL was

placed into a 100 mL volumetric flask, and

adjusted to the mark with a sample of known

concentration. The test solution for the

method

detection

limit and Limit

of

Quantitationwas made in ten replicates.

Test Sample Preparation

Preparation of Confirmation Limit of

Quantitation Solution

A total of 2 g of feed sample was weighed

and placed into a porcelain crucible, then

burned in a furnace at 550 °C for 4 h until all

the material turned to ash. The ash from the

combustion was allowed to cool, then 10 mL

of 3 M HCl solution was added, covered with

a watch glass, and heated for 10 minutes to

A total of 9 mL of standard solution with a

concentration of 10 mg/L was added to a 100

mL volumetric flask, then the volume of the

solution was adjusted to the mark using a

sample with a known concentration. The test

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solution

for

confirming

the

Limit

of

wavelength of 422.673 nm. The test results

Quantitation was prepared in ten replicates.

were

statistically

processed

and

then

compared with the acceptance criteria set by

AOAC. Data processing is calculated using

Microsoft Excel.

Linearity Test

The standard working solution of Ca (0; 2; 4;

8; 16; 20) mg/L that has been prepared was

measured using the ICP-OES 422.673 nm

instrument. The measurement results were

Limit of Detection Test

The prepared blank solution was analyzed

using ICP-OES at a wavelength of 422.673

nm. The obtained data were statistically

processed and subsequently compared with

the acceptance criteria established by AOAC.

All data processing and statistical calculation

were performed using Microsoft Excel.

statistically

processed

to

obtain

the

correlation value, slope, and intercept. The

analysis results are then compared with the

acceptance criteria set by AOAC. Data

processing is calculated using Microsoft

Excel.

Limit Test of Linearity Curve (LoL)

Method Detection Limit Test and Limit of

Quantitation

The working solution of the standard Ca with

the lowest concentration of 2 mg/L and the

highest concentration of 20 mg/L was

measured ten times using the ICP-OES

instrument at a wavelength of 422.673 nm.

The measurement results were statistically

processed to obtain the Fcalculated value.

The prepared solutions for the Method

Detection

Limit

(MDL)

and

Limit

of

Quantitation (LOQ) determination were

analyzed using an Inductively Coupled

Plasma-Optical Emission Spectrometer at a

wavelength of 422.673 nm. The reading data

The

Fcalculated

value

obtained

was

were

then

statistically

processed

to

compared with the acceptance criteria set by

AOAC. Data processing is calculated using

Microsoft Excel.

determine the relative standard deviation

(%RSD), percent recovery, signal-to-noise

ratio (S/N), as well as the LDM and LOQ

values. All evaluation results were compared

with the acceptance criteria established by

AOAC. All statistical calculation and data

processing were calculated using Microsoft

Excel.

Precision Test (Repeatability)

The precision test solution, which has been

prepared ten times, was measured using the

ICP-OES instrument at a wavelength of

422.673

statistically processed to determine the

precision value or relative standard

deviation (%RSD), and then compared with

the acceptance criteria set by AOAC. Data

processing is calculated using Microsoft

Excel.

nm.

The

test

results

were

Confirmation Limit of Quantitation Test

The confirmation solution for the Limit of

Quantitationthat has been prepared was

analyzed using the ICP-OES instrument at a

wavelength

of

422.673

nm.

The

measurement data were then statistically

analyzed to calculate the relative standard

deviation (%RSD) and recovery percentage.

Accuracy Test

These

values

are

then

evaluated

by

The accuracy test solution, which has been

prepared ten times, was then measured

comparing them against the acceptance

limits set by AOAC. Data processing is

calculated using Microsoft Excel.

using

the

ICP-OES

instrument

at

a

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Result and Discussion

the ground state to a higher energy level

[18]. Based on the method verification tests

that have been conducted, the results

include linearity parameters, Linearity Curve

Limit (LoL), Limit of Detection (LoD), Method

Detection Limit (MDL), Limit of Quantitation

(QL), Precision, and Accuracy. The results of

the method verification test for determining

calcium in animal feed can be seen in Table

1.

Method verification for calcium (Ca) content

analysis in animal feed was conducted using

the ICP-OES instrument. The basic principle

of this technique is the measurement of the

intensity of radiation emitted by atoms

during energy transitions due to excitation

[17]. ICP-OES operates based on the

characteristic of atoms that absorb light

energy and trigger electron excitation from

Table 1. Data Results of Verification of the Calcium Content Determination Method in Animal

Feed Using ICP-OES

No

Parameters

Results

Acceptance

Conditions

r ≥ 0.9950

Source

Conclusion

1

Linearity (r)

(0-20) mg/L

r = 0.9991

AOAC

(2020)

Meet The

Requirements

2

3

Limit Curve

Linearity (2 and

20) mg/L

Precision

(Repeatability)

0.03 < 3.18

F_count< F_table

AOAC

(2020)

Meet The

Requirements

%RSD = 2.18

%RSD ≤ ½

CV Horwitz

AOAC

(2020)

Meet The

Requirements

½ CV Horwitz =

7.92

(2.18≤ 7.92)

%Recovery =

(90-99)%

Accuracy

(Recovery)

%Recovery =

(80-115)%

AOAC

(2020)

Meet The

Requirements

4

Limit of

Detection (LoD)

Method

Detection Limit

(MDL) and Limit

of Quantitation

(LoQ)

0.0745 mg/L

Positive

Response

Reads

AOAC

(2020)

AOAC

(2020)

Meet The

Requirements

Meet The

5

6

Theoretical

MDL= 0.8079

mg/L

S/N = 9.08

%Recovery =

(86-113)%

LoQ = 0.9060

mg/L

positive

Requirements

2.5-10

%Recovery=

(80-115)%

Positive

Response

%RSD≤2/3

CV

Confirmation

Limit of

Quantitation

%RSD = 10,

2/3 CV Horwitz =

10.2

AOAC

(2020)

Meet The

Requirements

7

Horwitz

10≤10.2

Recovery = (86-

113)%

Recovery =

(80-115)%

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Linearity

correlation coefficient (r) value of 0.9991,

which is close to 1. This indicates a strong

Linearity describes the extent to which an

analytical method can produce a response

that is proportional to the concentration of

the analyte within a certain range. This linear

relationship is usually indicated by the

correlation coefficient (r) [19]. The linearity

calculation results are shown in Table 2, and

the calibration curve for the Ca standard

series obtained is shown in Figure 1.

and

proportional

linear

relationship

between the concentration of the element

Ca and the instrument response. The value

of this correlation coefficient also meets the

AOAC acceptance criteria, which is r ≥

0.9950. Linearity evaluation was performed

using six calibration points (0.00–20.00

mg/L), and the regression results showed

excellent linearity for Ca determination

using ICP-OES. The regression model yielded

a coefficient of determination (R²) value of

0.99842, indicating that over 99% of the

variation in the optical signal can be

explained by changes in Ca concentration.

Based on the graph, the linearity test results

show that the intensity of the calcium signal

is directly proportional to the calcium

concentration. The regression equation

obtained is y = 293,320x – 163,402 with a

Table 2. The linearity calculation results

Concentration

Intensity

No

of Ca (mg/L)

(Xi)

(Xi)*(Yi)

Xi²

Yi²

(Yi)

0.0

0

1

0.00

2

3

4

5

2.00

4.00

8.00

16.0

20.00

343,348.52

931,870.22

2,124,684.91

4,559,671.64

5,726,023.29

13,685,598.58

686,697.04

372,7480.88

16,997,479.28

72,954,746.24

114,520,465.8

208,886,869.2

293,320.25

4

16

64

256

400

740

117,888,206,186.19

868,382,106,922.85

451,428,596,6781.71

20,790,605,464,620.30

32,787,342,717,622.40

59,078,504,462,133.50

6

Amount

Slope (b)

Intercept

(a)

50.00

–163,402.32

r

0.9991

0.9984

R²

(Sy/x)

104,897.86

In

linear

regression

analysis,

the

between two datasets [21]. An ideal

relationship occurs when the intercept value

approaches zero and r approaches +1 or −1,

relationship between variables is expressed

through the equation y = bx + a, where b is

the slope of the line, a is the intercept, x

represents the analyte concentration, and y

denotes the instrument response [20]. The

correlation coefficient (r) is used to assess

the strength of the linear relationship

depending

correlation.

on

the

direction

of

the

From the obtained curve, the intercept value

(a) is -163,402.32. An intercept close to zero

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Chempublish Journal, 9(2) 2025, 313-329

indicates that the instrument does not

provide a signal when a blank solution is

tested [22]. However, the presence of

interference, noise, contamination, or other

instrument effects often causes a signal to

still be read even for a blank. A slope value

(b) of 293,320.25 indicates the sensitivity

level of the method. The larger the slope

value, the higher the method's sensitivity to

the analyte being tested [23].

Figure 1. Curve of Calcium Concentration vs. Intensity

Residual analysis showed random

a

statistical parameters confirm that the

calibration curve satisfies the linearity

requirements recommended by AOAC and

ISO/IEC 17025, demonstrating that the

method is suitable for the determination of

Ca in animal feed.

distribution around the zero line with no

observable systematic pattern, indicating

absence of lack-of-fit and confirming that

the linear model is adequate over the tested

concentration range. The standard error of

regression

demonstrates

relationship between analytical intensity

(Sy/x)

value

of

104,897.86

Limit Curve Linearity

good

precision

in the

The linearity limit of the curve was measured

by comparing with the standard solutions of

Ca using concentrations 2 mg/L and 20 mg/L,

each 10 times. The results of the two

standard deviations obtained are compared.

The data limits for the linearity curve and the

and

confidence

analyte

concentration.

interval for

The

the

95%

slope

(277,123.40–309,517.10) indicates stable

instrument sensitivity, while the confidence

interval for the intercept (–343,277.29 to

16,472.63) suggests that the intercept does

not significantly differ from zero, indicating

the absence of systematic bias at the zero

F-table

are

shown

in

Table

3.

The

comparison of the standard deviation of the

data from the repeated tests is in the form of

F-count. The calculated F count is 0.018.

Based on degrees of freedom df1=df2=n-1

and a confidence level of 95% (α = 0.05), the

value of F-tabel is obtained as 3.18. From the

calculation data, it is known that F-count < F-

concentration

level.

Although

measurements were performed without

replication at each concentration level, the

calibration design remains appropriate for

initial method verification. Overall, these

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table, so H₀is accepted, meaning the

precision level of the lowest working solution

intensity is not significantly different from

the precision level of the highest working

solution intensity [24]. Based on the results

obtained, with a confidence level of 95% (α =

0.05), 2 mg/L – 20 mg/L is a linear regression

and 20 mg/L is the highest concentration

detectable by the instrument.

Table 3. Data limits for linearity curve and F-Table

Intensity

Sample

Lowest Standard

Highest Standards

(20 mg/L)

(2 mg/L)

1

2

3

4

5

6

7

8

350,226.9894

332,253.1689

349,590.3771

350,098.3865

347,700.5273

332775,5831

333,089.9671

343,347.0374

347,605.1231

338,383.0410

8,935.336

5,773,543.8410

5,741,227.0290

5,697,047.2310

5,579,630.9190

5,658,376.0490

5,652,233.3640

5,689,108.3010

5,658,277.0960

5,625,120.0700

5,624,015.7550

51,372.977

9

10

SD

SD²

79,840,222.0

2,639,182,775.4

F_count

F_table

(df = n-1, α = 5%)

0.018

3.18

Precision (Repeatability)

objective comparison of method precision

across different concentration levels, with

lower values indicating higher consistency

and analytical reliability. The experimental

results yielded a %RSD of 2.18%, which was

evaluated against the Horwitz coefficient of

variation (CV) of 7.92%. The Horwitz CV,

derived from an empirical model, serves as a

widely accepted benchmark for assessing

acceptable precision in chemical analysis.

Precision testing is an essential parameter in

analytical method validation, intended to

evaluate the degree of agreement among

repeated measurements obtained under

identical analytical conditions. Precision

reflects the magnitude of random errors in

an analytical procedure and provides insight

into the short-term reproducibility of the

method. In this study, precision was

assessed through a repeatability test, in

which replicate analyses were performed

using the same analyst, instrumentation,

According

established by AOAC, a method meets

repeatability requirements when the

to

the

acceptance

criteria

observed %RSD does not exceed 0.5 × CV

Horwitz.

reagents,

and

experimental

conditions

within a limited time frame [25].

In this case, the obtained %RSD was

significantly lower than the allowable limit

(3.96%), confirming that the analytical

The repeatability of the method was

expressed as the relative standard deviation

(%RSD),

a

statistical

parameter

that

method

demonstrates

satisfactory

describes data dispersion relative to the

mean value. The use of %RSD allows for

repeatability. These results indicate that

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random

variability

is

well

controlled,

RSD ≤ 1%

: very accurate

(1)

(2)

(3)

(4)

supporting the method’s reliability and

suitability for routine quantitative analysis

[26]. The relative standard deviation value

obtained from the precision test indicates

that the precision test results fall into the

medium accuracy category.

1% < RSD ≤ 2% : high accuracy

2% < RSD ≤ 5% : moderate accuracy

RSD ≥ 5 %

: low accuracy

Table 4. Precision test data and calculations

C

V

C

Actual

Test

Sampel

(mg/L)

Spike

(mg/L)

Spike

(mL)

Sampel

(mL)

Target

(mg/L)

Intensity

Concentration

of Ca (mg/L)

1.022381

1.03481

1

2

3

4

5

6

7

8

9

132,409.6

136,044.6

140,934.6

145,760.1

145,603.9

149,725.7

143,037.5

151,478

1.051531

1.068031

1.067497

1.08159

1.058721

1.087582

1.094452

1.080174

10.64677

1.064677

0.023

0.6600

10

5

95

1,1270

153,487.3

149,311.4

10

Amount

Average

SD

% RSD

0,5 CV Horwitz

2.18

7.92

Accuracy (Recovery)

using a standard solution with a known

concentration. The results of the accuracy

test and the corresponding calculations are

presented in Table 5.

Accuracy is a key parameter in analytical

method

closeness

validation

of agreement

that

describes

between

the

measured value and the true or accepted

reference value of an analyte [27]. It reflects

the trueness of an analytical method and

indicates the presence of systematic error. In

quantitative analysis, accuracy is commonly

evaluated through recovery studies, in which

a known amount of analyte standard is

spiked into the sample matrix and analyzed

using the proposed analytical method. This

approach allows evaluation of matrix effects,

analyte loss during sample preparation, and

The obtained %Recovery values fall within

the acceptable limits recommended by the

AOAC guidelines for analytical method

validation, indicating that the method

produces reliable and unbiased results.

Based on ten replicate analyses, the

%Recovery values ranged from 90% to 99%,

with an average recovery of 94%. These

results comply with the acceptance criteria

established by AOAC (2002), which specify an

acceptable recovery range of 80–115% for

quantitative analytical methods. Therefore,

the method.

potential

analytical

bias.

Accuracy

is

generally expressed as the percentage

recovery (%Recovery) of the added analyte

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Table 5. Accuracy Data and Calculation

Actual

Concentration

C

V

C

Test

Sampel

(mg/L)

Spike

(mg/L)

Spike

(mL)

Sampel

(mL)

Target

(mg/L)

Intensity

% Recovery

Calcium

(mg/L)

1.0224

1.0348

1.1045

1.0515

1.1163

1.0675

1.0945

1.0587

1.0008

1.1258

1.0677

1

2

3

4

5

6

7

8

132,409.6

136,044.6

156,432.9

140,934.6

159,879.9

145,603.9

153,487.3

143,037.5

126,101.8

162,660.6

90

91

98

93

99

94

97

93

88

99

94

0.6600

10

5

95

1.1270

9

10

Average

Accuracy Range Value

Acceptance Conditions

(90-99) %

(80-115) %

Limit of Detection (LoD)

parameter reflects the minimum sensitivity

of an analytical method. The LoD can be

determined by measuring blank samples,

which are matrices free of the analyte, to

confirm that the instrument response

originates from the presence of the analyte

rather than background noise [29].

The data and LoD calculations are presented

in Table 6. The limit of detection is defined as

the lowest concentration of an analyte in a

sample that produces a signal significantly

different from that of the blank [28]. This

Table 6. Data and LoD Calculations

2

Test

1

2

3

4

5

6

7

Intensity

17,400.6287

16,400.4184

3,920.1495

821.1895

1,259.3401

2,038.0774

338.3124

285.8692

235.6276

12,393.7679

3.0223

Concentration (xi) (mg/L)

(xi-

)

0.3443

0.3408

0.2966

0.2856

0.2872

0.2899

0.2839

0.2837

0.2836

0.3022

0.04207

0.03853

-0.00562

-0.01658

-0.01503

-0.01228

-0.01829

-0.01848

-0.01866

0.02435

0.0018

0.0015

0.0001

0.0003

0.0002

0.0003

0.0006

0.0056

8

9

10

Amount

Average (mg/L)

SD

0.3022

0.0248

LoD (mg/L)

0.0745

323

Syafrinal., et al.

Chempublish Journal, 9(2) 2025, 313-329

In this study, the theoretical LoD value

obtained was 0.0745 mg/L, representing the

lowest concentration of Ca that could be

reliably detected by the instrument. The LoD

evaluation met the acceptance criteria

established by AOAC (2002), as it produced a

sample

that can

still

be

determined

quantitatively with acceptable levels of

precision and accuracy, under agreed-upon

test conditions [31]. The LOQ value and

method detection limit were determined by

measuring the prepared solutions using the

analyte addition (spiking) technique. Data

and Calculations for the Detection Limit of

the Method and Limit of Quantitationare

shown in Table 7.

positive

and

distinguishable

analytical

response

Method Detection Limit

Determining the method's detection limit

reflects both the laboratory's capabilities

and limitations in applying an analytical

In this test, the theoretical concentration

values for MDL and LoQ were obtained. The

method's limit of detection is acceptable if

the data from the test repetitions meet

several acceptances following limit formula

5-9.

method

to

detect

analytes

at

low

concentrations. The method's limit of

detection is defined as the concentration of

an analyte that can be detected thru all

stages of the analysis procedure with 99%

confidence, where the signal produced can

be clearly distinguished from the blank

signal [30]. Limit of Quantitation (LOQ), often

referred to as the reporting limit, is the

lowest concentration of an analyte in a

%RSD < 0,67 Horwitz

(5)

(6)

(7)

(8)

(9)

% Recovery = 80-115 % (AOAC, 2002)

The signal to noise ratio (S/N) = 2,5-10

MDL < Spike < 10MDL

LoQ ≤ BML

Table 7. Data and Calculation of Method Detection Limit and Limit of Quantitation

C

V

C

Recovery

(%)

Test

Sampel

(mg/L)

Spike

(mg/L)

Sampel

(mL)

Spike

(mL)

Target

(mg/L)

Intensity

Calcium

(mg/L)

2.9500

2.8900

2.3000

2.2800

2.6000

2.6500

2.2500

2.8900

2.8600

2.3500

26.0200

2.6020

0.2864

0.0906

9.08

1

9,762,972 ,568

1,006,484,546

1,047,067,863

1,092,602,128

1,129,398,123

1,125,396,591

1,150,724,794

1,179,239,004

1,554,535,717

2,011,566,989

113 %

111%

88%

2

3

4

87%

5

100%

102%

86%

0.6600

20

90

10

2.5940

6

7

8

111%

110%

90%

9

10

Amount

Average

SD

SD’ SD /√푛

S/N

MDL theoretical (mg/L)

10 MDL (mg/L)

0.8079

8.079

LoQ theoretical (mg/L)

0.906

The method detection limit (MDL) must

satisfy acceptable precision and accuracy

criteria. Precision is commonly expressed as

the coefficient of variation, also referred to

as the relative standard deviation (%RSD).

The

%RSD

obtained

from

repeated

324

Syafrinal., et al.

Chempublish Journal, 9(2) 2025, 313-329

measurements

process should not exceed 0.67 times the

Horwitz coefficient of variation (CV) value

during

the

verification

baseline method limit (BML) with the

corresponding limit of quantitation (LoQ).

This approach is based on the established

working range of the method, in which the

lower limit is defined by the LoQ and the

upper limit by the limit of linearity (LoL).

Consequently, the LoQ serves as a critical

parameter for determining the applicability

and reliability of an analytical method for

quantitative measurements [34].

[32].

In

addition,

the

recovery

test

(%Recovery) results must fall within the

acceptable range of 80–115%.

The signal-to-noise ratio (S/N) is used to

assess the level of random error that may

occur during the testing process and to

estimate the precision of repeated test

results which is calculated using Microsoft

Excel. If the S/N ratio is less than 2.5, this

indicates that the random variation in the

replicates is quite high and may cause the

method detection limit (MDL) to be large. In

such conditions, the concentration of the

added analyte (spike) needs to be increased

to produce a stronger signal. Conversely, if

the S/N exceeds 10, it means the amount of

Based on the limit of quantitation test

results, the theoretical LoQ was determined

to be 0.9060 mg/L, representing the lowest

analyte concentration that can be quantified

with acceptable precision and accuracy. This

theoretical value required confirmation to

ensure

compliance

with

method

performance requirements. Therefore, a

LoQ confirmation test was conducted. The

results demonstrated that the method met

the required precision and accuracy criteria

and complied with the acceptance standards

established by the AOAC (2002), confirming

the suitability of the method for quantitative

analysis

analyte

added

is

too

high,

so

its

concentration needs to be reduced to

remain within the ideal working range [33].

When determining the method detection

limit (MDL), the choice of spike concentration

must be made carefully to ensure the results

obtained are within an acceptable range. The

precision level in determining the MDL is

highly influenced by the concentration of the

spike used. If the obtained MDL value

exceeds the spike level, it will be statistically

difficult to distinguish between the spike

value and the blank value, resulting in low

precision. Therefore, the determination of

spike levels should consider the lower limit

of the concentration range being verified, as

well as follow the recommended ratio, which

is MDL : LoQ = 4 : 10.

Confirmation Limit of Quantitation (LoQ)

The

data

and

calculations

for

the

confirmation of the limit of quantitation

(LoQ) are presented in Table 8. Based on the

quantitation test results, the theoretical LoQ

was determined to be 0.9000 mg/L, which

represents the lowest analyte concentration

that can be quantified reliably and therefore

requires confirmation to ensure that the

method meets the required precision and

accuracy criteria [35]. LoQ confirmation was

carried out by analyzing a calcium standard

solution with a concentration of 10 mg/L

When the method detection limit (MDL)

meets the required statistical acceptance

using

ICP-OES.

The

precision

of

the

criteria,

the

obtained

value

may

be

measurements was evaluated in terms of

the relative standard deviation (%RSD), and

the confirmation test yielded an %RSD value

of 10%. This value satisfies the applicable

acceptance criterion, namely %RSD ≤ 2/3 CV

compared with relevant environmental

laboratory quality standards. In routine

practice, laboratories that have verified their

analytical methods may also compare the

325

Syafrinal., et al.

Chempublish Journal, 9(2) 2025, 313-329

Horwitz,

indicating

that

the

analytical

LoQ is suitable for reliable quantitative

method demonstrates adequate precision at

the quantitation limit and that the confirmed

determination

concentration levels.

of

calcium

at

low

Table 8. Data and Calculation for Confirmation Limit of Quantitation

C^a

Sample

(mg/L)

C

C Ca

actual

(mg/L)

1.3014

1.2876

1.3252

1.3546

1.3541

1.3795

1.3196

1.2821

1.7114

1.5635

13.8790

1.3879

0.1395

10.0

C

V^c

(mL)

V^d

(mL)

Recovery

Test

Target

(mg/L)

Intensity

xi-x

̅

(xi-x̅)2

(mg/L)

(%)

1

2

3

4

5

6

7

8

371,290

367,644

377,557

385,331

385,191

391,908

376,094

366,178

479,541

440,481

86

85

88

90

91

87

85

114

104

-0,0864

-0,1002

-0,0627

-0,0333

-0,0338

-0,0083

-0,0682

0,1058

0,3234

0,1755

0.0075

0.0101

0.0039

0.0011

0.0001

0.0047

0.0112

0.1046

0.308

0.6600

10

91

9

1.5006

9

10

Amount

Average

SD

% RSD

% Recovery

2/3 CV Horwitz

(85-114) %

10.2

Conclusions

animal feed, both for quality control and

regulatory compliance purposes. However,

this study has certain limitations, particularly

The verification results demonstrate that the

ICP-OES method based on SNI 3148.2:2009

provides adequate analytical performance

for the determination of calcium in livestock

the

absence

of

a

comprehensive

evaluation.

measurement

uncertainty

Therefore, further research is recommended

to assess the influence of different feed

feed

matrices.

The

method

exhibited

excellent linearity, stable precision, and

satisfactory accuracy within the acceptance

limits recommended by the AOAC, indicating

a consistent and proportional instrument

matrices

and

to

perform

structured

measurement uncertainty calculations in

accordance with ISO/IEC 17025. Addressing

these aspects would further strengthen

method validity and broaden its application

in industrial-scale laboratory testing.

response

to

variations

in

calcium

concentration. Furthermore, the low values

of the limit of detection (LOD), limit of

quantitation (LoQ), and method detection

limit (MDL) confirm that the method is

sufficiently sensitive to detect and quantify

Acknowledgement

This research supported by the Ministry of

Industry Affairs Republic Indonesia through

collaboration research by lecturers from

Polytechnic ATI Padang.

calcium

at

low

concentration

levels,

supporting its applicability for feed quality

testing across diverse matrix compositions.

Overall, the verification confirms that ICP-

OES is a reliable and suitable analytical

technique for routine calcium analysis in

Author Contributions

326

Syafrinal., et al.

Chempublish Journal, 9(2) 2025, 313-329

The contribution of each author to this

article is : "Conceptualization, Syafrinal and

Nurmaliza.; Methodology, Syafrinal and

2023.

391–446.

https://doi.org/10.1007/978-981-19-

4150-4_11

Nurmaliza;

Software,

Hafnimardiyanti.;

[4]. Wu, G. Management of metabolic

Validation, Syafrinal., Nurmaliza and Selfa

Dewati Samah.; Formal Analysis, Pevi Riani.;

Investigation, Renny Futeri.; Resources,

Syafrinal.; Data Curation, Nurmaliza.; Writing

– Original Draft Preparation, Syafrinal and

Nurmaliza; Writing – Review & Editing,

Syafrinal.; Visualization, Hafnimardiyanti.;

disorders

diseases)

ruminant

Agriculture.

(including

ruminant

animals.

2020.

metabolic

in

and

In

non-

Animal

471–491).

https://doi.org/10.1016/B978-0-12-

817052-6.00027-6

[5]. Carter, S., Clough, R., Fisher, A., Gibson,

B., Russell, B., & Waack, J. Atomic

Supervision,

Administration,

Pevi

Selfa

Riani.;

Project

Dewati

Samah.;

spectrometry

update:

Review

of

Funding Acquisition, Renny Futeri.

advances in the analysis of metals,

chemicals and materials. Journal of

Analytical Atomic Spectrometry, 34(11):

Conflict of Interest

2159–2216.

2019.

The authors declare that there is no conflict

of interest regarding the publication of this

paper.

https://doi.org/10.1039/C9JA90058F

[6]. Khan, S. R., Sharma, B., Chawla, P. A., &

Bhatia, R. Inductively coupled plasma

optical emission spectrometry (ICP-

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for elemental analysis. Food Analytical

Ethical Standards

This article does not contain any studies

involving human or animal subjects.

Methods,

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666–688.

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