ISSN 2186-9138 / JFRLNE 002
Analysis of Vitamins ~ Microbiological assay and HPLC ~ 1/5

JFRL News Vol.6 No. 6 Feb. 2018
Analysis of Vitamins~ Microbiological assay and HPLC ~Introduction
Vitamins are nutrients essential for the normal functioning of the body and are organic compounds
that must be taken in from the diet because they cannot be synthesized in the body or even when synthesized,
their quantities are insufficient to meet the physiological demand. Currently, there are thirteen vitamins,
which are broadly divided into two groups; four fat-soluble vitamins and nine water-soluble vitamins. In
recent years, there has been increasing interest in health-consciousness and the functionality of foods. The
contents of nutritional and functional ingredients, including vitamins, has become more important than ever.
Therefore, accurate analytical data of these ingredients should be controlled and properly reflected onlabeling. The Analytical methods for vitamins in the Food Labeling Standards 1) in Japan are mainly basedon microbiological assays and high-performance liquid chromatography (hereinafter referred to as HPLC).
We analyzes several B-vitamins (vitamin B6, vitamin B12, folic acid, pantothenic acid, niacin, and biotin)
using microbiological assay and HPLC. In this article, we introduce the characteristics of both analyticalmethods.

Analysis method(1) Microbiological assay
Principle of the method
Microbiological assay is a method to quantify microorganisms that requires the vitamin of purpose
as an essential nutrient. It quantifies vitamins by measuring the degree of growth when they are cultured in a
medium without the vitamin.
The summary of the test method is as follows;.
1) Samples are weighed and then, pressurized extraction and enzymatic treatment are conducted to separate
proteins and sugars which are converted into vitamins. These can be used by organisms for growth, andsample solutions are obtained.
2) Add the standard solution or sample solution and the test organism solution to the culture medium, andincubate in test tubes or microplates.
3) After incubation, measure the turbidity (600 nm) of the culture solution. The more vitamins are containedin the standard solution or sample solution, the greater the turbidity will be because the test organism grows
and the culture solution becomes turbid (Figure 1).

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Absorbance (600 nm)

Analysis of Vitamins ~ Microbiological assay and HPLC ~

2/5

0.3
0.2
0.1
002

4

6

Niacin amount (ng)Figure 1 Example of a calibration curve for niacin.
Characteristics of the method
Microbiological assay is a highly sensitive method that can measure the targeted vitamin B to alow concentration range without being affected by impurities in the sample. It is also possible tosimultaneously measure naturally originating substances and ingredients with the same activity. For example,vitamin B6 can measure pyridoxine, pyridoxal, pyridoxamine, and their phosphate forms at once and quantify
the total amount of vitamin B6. On the other hand, the measurement range of the calibration curve is narrow,and the growth of the test organism may affect the results. It is also time consuming, since culture of the test
organism is required. The handling of test organism requires a certain level of know-how, and is therefore amore difficult analytical method than HPLC. Other disadvantages are difficulties in separating and measuring
each active ingredient.
(2) HPLC
Principle of the method
The target vitamins are extracted from the samples, separated from other impurities by HPLC
columns, then detected and quantified by an ultraviolet-visible spectrophotometer or a fluorescence detector.
This method is applied to samples with high levels of added vitamins so it does not require complex
pretreatment.
Characteristics of the method
HPLC method is a reliable method which can obtain high precision data in a short time. It is also
possible to measure the target substance individually. For example, it can measure only pyridoxine as vitamin
B6. On the other hand, the detection sensitivity is less than that of microbiological assays, and it is notpossible to measure total of natural vitamins such as analogs and various conjugates, simultaneously.
Additionally, because of its low sensitivity and high precision character, impurities in the samples cause
difficulties to obtain the results.

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Comparisons between microbiological assays and HPLCThe Food Labeling Standards state examples of HPLC procedures for six vitamins (vitamin B6,vitamin B12, folic acid, pantothenic acid, niacin, and biotin) for which microbiological assay is the mainstay,and samples with high contents, such as health foods and vitamin preparations, can be analyzed by HPLC.
Vitamin B6, folic acid, pantothenic acid, and niacin in these samples can generally be analyzed by HPLC,
but vitamin B12 and biotin are generally contained in low quantities in foods, which makes the kinds ofapplicable sample limited. Table-1 compares the microbiological assay with HPLC
Table-1 Compares between microbiological assays and HPLC
Microbiological assay
Measurement sensitivity

HPLC

Measurable from low to highMeasurable

concentration range

concentration range

Narrow

Wide

Analysis time

Require time

Can be analyzed in a short time

Analytical precision

Be slightly inferior

PreciseTest procedures

Require pretreatment

No

Measurement range of theinthehighcalibration curvecomplexpretreatment

required
Influence of impurities
Substances measured

Low possibility to be affected

Impurities in the sample may

by impurities in the sample

preclude analysis.

Measured as total amountIndividual

including conjugated vitamins

target substances

measurementofand analogs
Applied sample

General foods (fresh foods,

Health foods supplemented

processed foods, and healthwith vitamins for ingestion

foods such as supplements),

and reinforcement purposes

feeds(supplements, etc.) andvitamin preparations(premixes, etc.)

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Analysis of Vitamins ~ Microbiological assay and HPLC ~4/5[1] Quantitation limit
Microbiological assays are very sensitive for detection. Our quantitation limit of HPLC method is
0.5 mg/100 g, but microbiological assays can measure from 0.03 μg/100 g to 0.05 mg/100 g. It is 10 to 10,000
times of HPLC (Table-2).
Table-2 Quantitation limits
Composition measured

Microbiological assay

HPLC

Vitamin B60.003 mg/100g

Vitamin B120.03 μg/100g

Folic acid

1 μg /100g

0.5 mg/100g

Pantothenic acid,0.05 mg/100g(500 μg/100g)

Niacin0.03 mg/100g

Biotin0.3 μg/100g[2] Correlation between the two assays
Table-3 shows the values of vitamin B6 in three dietary supplements when analyzed by both
microbiological assay and HPLC. Results show samples with high concentrations of vitamins have similarvalues using both methods.
Table-3 Comparable data of vitamin B6 values by microbiological assay and HPLC
SampleTablet A

Microbiological assay

HPLC ※1(mg/100g)(mg/100g)

64.9

68.9

Tablet B

1,140

1,070

Tablet C

6,850

6,810※1 Values converted from pyridoxine hydrochloride to vitamin B6. Conversion factor ;0.8227[3] Analytical precision
Analytical precision of microbiological assay and HPLC is shown as the relative standard
deviations (%) of the measured values (Table-4). Supplements added with high concentrations of eachvitamin were analyzed 10-20 times, and their relative standard deviations (standard deviation/mean × 100)
were calculated. The precision of HPLC analysis is better for all vitamins.

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Analysis of Vitamins ~ Microbiological assay and HPLC ~5/5

Table-4 Analytical precision [Relative standard deviation (%) of measurements].
Measurement component(concentration level)

Microbiological assay

HPLC

Vitamin B6(1,000 mg/100 g)

10.1

2.69

Folic acid(50 mg/100 g)

7.68

2.12

Pantothenic acid

(2,000 mg/100 g)

5.62

3.01

Niacin(4,000 mg/100 g)

3.11

1.71

Closing
The microbiological assay has excellent detection sensitivity and enables the measurement of target
vitamins in low concentration range, and enables the determination of the total amount of target vitamins,
including naturally originating substances and analogs. However, the measured values have low
reproducibility because they are affected by the growth of organisms, and the analytical precision is inferior
to that of HPLC. The lacking reproducibility of the measured values may be due to changes in the conditionsof the test organism, culture media, or seasonal variation. On the other hand, HPLC can be analyzed withhigh precision in a short time. It is a very useful analytical method when we want to analyze the amounts ofvitamins in supplements and premixes which are supplemented with vitamins for fortification purpose as
well as the change in the amount during their storage.
Vitamin B12 and biotin, which are not listed in Table-4, can also be analyzed depending on the amount added
and the matrix of the sample.
In Japan, the Food Labeling Standard shows microbiological assay prior to HPLC for vitamin B6,vitamin B12, folic acid, pantothenic acid, and biotin. The official methods in the Food Labeling Standards isused for sampling inspections by the authorities. It is not mandatory to use official methods to obtain the datafor labelling. You can use suitable method according to the sample matrices. Microbiological assay isapplicable to various matrices of common food samples and able to detect low level of vitamins. For samples
with added vitamins and when formulation is obvious, HPLC is applicable.
We are looking forward you contact regarding vitamin analysis.
References
1) Consumer Affairs Agency: Food Labeling Standards (Digestion Table No. 139 of March 30, 2015)
Attachment Analytical Method of Nutrient Ingredients, etc.

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