Vitamin testing solutions
from R-Biopharm

Determination of natural and/or synthetic vitamins,
single or multi-vitamin testing options

More information:
https://r-b.io/vitamins_EN

ELISA, IAC, microbiological test kits or reusable cartridges

Global availability for test kits and equipment

R-Biopharm • An der neuen Bergstraße 17, 64297 Darmstadt, Germany • E-mail: info@r-biopharm.de • food.r-biopharm.com

Vitamin testing solutions

What are vitamins?
Vitamins are essential nutrients for humandevelopment and metabolism. Vitamins can beseparated into water soluble vitamins (e.g. vitamin
C and the vitamins of the B group) and fat soluble

vitamins (e.g. vitamin A, D, E and K). The humanbody cannot synthetize vitamins (except vitamin K
and D). Therefore, vitamins have to be provided byfood or supplements (see figure 1).

Infant formula

Supplements

Baby food

Flour

Breakfast cereals

Beverages

01/2023, page 2

Figure 1: Frequently tested samples for vitamins are infant formula, baby food, nutritional supplements, flour,
breakfast cereal and beverages.

Vitamin testing solutions

Why are food products tested for vitamins?
Accurate measurement of vitamin levels in foods
is critical to comply with labelling requirements,
product quality and internal product specifications.
Additionally, by conducting regular testingproduction errors (e.g. problems during theaddition of vitamin pre-mixes) are recognized early.
Vitamins are susceptible to degradation, meaningthat the levels in the food’s formulation may not be
the same as what is present in the final product due
to heat, UV light, incorrect pH and certain metals.
Hence, it is necessary that manufacturers as well as
governmental labs control vitamin contents.

Quantify added or natural vitamins?
Food can contain natural as well as synthetic
vitamins (see table 1). Fortification with synthetic
vitamins is usually done by adding pre-mixes into
the product. Knowing the form(s) of the vitaminin a food is critical to ensure the correct sample
preparation and choosing the respective analyticalmethod.

Table 1: Some examples for natural and/or synthetic vitamin forms.

01/2023, page 3

Vitamin
B1

Thiamin, thiamin hydrochloride, thiamin mononitrate

B2

Riboflavin, riboflavin-5’-phosphate

B3

Niacin, niacinamide

B5

Pantothenic acid, calcium pantothenate

B6

Pyridoxine, pyridoxamine, pyridoxal, pyridoxine hydrochloride

B7

BiotinB9

Folic acid, multiple naturally-occurring folates

B12

Hydroxocobalamin, methylcobalamin, cyanocobalaminC

Ascorbic acid, deoxyascorbic acid, multiple synthetic forms

Vitamin testing solutions

What analytical methods are offered by R-Biopharm?
R-Biopharm AG offers different test kits for the detection of water soluble vitamins in food and supplements.

VitaFast®
Microbiological tests• Samples with an added or natural vitamin content
can be analyzed• Method in conformity with official guidelines(§ 64 of the German Food & Feed Act, AOAC)• AOAC-RI certification for some VitaFast® tests• Ready-to-use reagents and standards
for 96 determinations• Results available within 24 - 48 hours

RIDASCREEN®
ELISA• Determination of total vitamin B12 content• Determination of added folic acid vitamin• One sample preparation procedure andone identical sample buffer for
RIDASCREEN®FAST Vitamin B12 and Folic Acid
• Results within 1 - 2 h• Ideal for process control

EASI-EXTRACT®
Immunoaffinity columns• Isolation and concentration of the vitamin

01/2023, page 4• Pigments and interfering compounds are removed• High recovery and low coefficient of variation• Products referenced in AOAC Official Final Action
Methods• Unique multi-vitamin immunoaffinity column for thesimultaneous detection of vitamin B12, folic acidand biotin

Vitamin testing solutions

What do the different analytical methods detect?
The RIDASCREEN®FAST Vitamin brand contains two
different ELISAs, which are designed for vitamintesting. The antigen-antibody reaction of therespective ELISA is evaluated by a microtiterplate
photometer. The immunoassay kit is suitable formulti-vitamin tablets, capsules, multi-vitamin juices,multi-vitamin jam, grain products, multivitamin
sweets etc. RIDASCREEN®FAST Vitamin B12(Art. No. R2103) detects total vitamin B12 and
RIDASCREEN®FAST Folic acid (Art. No. R3203)
detects added synthetic folic acid.
The brand VitaFast® includes different
microbiological test kits, which detect added or

natural vitamins. The wells of the microtiter plate arecoated with lyophilized microorganisms. The growthof microorganisms is measured turbidimetricallywith a microtiterplate reader. Different vitaminparameter are available for all water-soluble
vitamins.
HPLC/LC-MS/MS in combination withimmunoaffinity columns quantifies total vitamincontent (vitamin B12 and biotin) or added vitamincontent (folic acid). EASI-EXTRACT® MULTI-VIT B(LGE) allows to measure multiple vitamins (biotin,
vitamin B12 and folic acid) simultaneously.

Table 2: Comparison of the vitamin testing methods for the analysis of vitamin B12.
RIDASCREEN®FAST

VitaFast®

EASI-EXTRACT®

ELISAMicrobiological test

Immunoaffinity column
prior to HPLC or
LC-MS/MS detection

Vitamin B12 total content,
Folic acid added content

Added or total vitamincontent depending on
sample preparation

Vitamin B12 total content,
biotin total content or folicacid added content

Easy to use, no toxic
chemicals for total
vitamin B12

AOAC-RI

AOAC-OMA

Time to result(e.g. 10 samples B12)

65 min

48 h

7.5 h

Hands-on time(e.g. 10 samples B12)

40 min4h

4.5 hMethodVitamins measured

01/2023, page 5

Benefits

Vitamin testing solutions

What equipment is required for the different vitamin test kits?
Depending on the analytical method, different
equipment is needed. The graphic below combines
the method with the necessary equipment for three
Lab starting with vitamin analysis(minimal equipment)• VitaFast®
• RIDASCREEN®FAST ELISA

MTP reader• RIDA®ABSORBANCE 96

different lab sizes (starting lab, lab with HPLC, fully
automized lab).

Lab equipped with HPLC
• VitaFast®
• RIDASCREEN®FAST ELISA• EASI-EXTRACT® immunoaffinity
columns

MTP reader• RIDA®ABSORBANCE 96

Accessories
VitaFast® Spiking Standard Folic acid,
Vitamin B12, Biotin and Pantothenic acidt (min)HPLC
Accessories
VitaFast® Spiking Standard Folic acid, Vitamin B12, Biotin and Pantothenic acid

Fully automized lab• EASI-EXTRACT® immunoaffinity
columns• EASI-EXTRACT® immunoaffinity
columns

IMMUNOPREP®
ONLINE cartridges

HPLC

Intensityt (min)LC-MS/MS

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atog

ram

Re

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01/2023, page 6

Mas

tru
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Automated system for use with HPLCor LC-MS/MS• CHRONECT Symbiosis RIDA®CREST

Vitamin testing solutions

What equipment is available from R-Biopharm AG?
For the evaluation of the ELISA and microbiological test kits R-Biopharm AG offers the RIDA®Absorbance 96(Art. No. ZRA96FF). The RIDA®CREST could be used for immunoaffinity cartridges for vitamin B12.

RIDA®ABSORBANCE 96
Absorbance reader
Innovative microtiter plate photometer including
RIDASOFT® Win.NET software

CHRONECT Symbiosis RIDA®CREST

01/2023, page 7

UHPLC system for automated, online clean-up using
the IMMUNONPREP® ONLINE immumoaffinity
cartridges

Vitamin testing solutions

What products have been externally validated?
The AOAC-PTM program certifies that a method
performs according to the manufacturer’s
documented claims. The manufacturer submits its
internal validation. The AOAC accepts the data, andan independent lab carries out verification with
exemplary samples from the internal validation(typically 2 or 3 matrices are verified). The reportfrom the independent lab is reviewed by the AOAC
experts.

Methods having an AOAC-OMA validation have been
more rigorously tested than methods having only
an AOAC-PTM certificate. A collaborative study witha minimum of 5 test materials and at least
8 independent labs needs to be carried out andstrict method performance criteria need to be met.

Table 3: Overview about the approvals of the vitamin test kits.
VitaFast®

EASI-EXTRACT®

RIDASCREEN®

IMMUNOPREP®

Microbiological /enzymatic tests

Immunaffinity
columns

ELISA

Onlineimmunoaffinity
cartridges

Folic Acid

AOAC-PTM



Vitamin B12 (Cyanocobalamin)

AOAC-PTM

AOAC-OMAVitamin B7 (Biotin)

AOAC-PTM

AOAC-OMA

Vitamin B3 (Niacin)Pantothenic Acid

AOAC-PTM

Vitamins

Vitamin B1 (Thiamin)Vitamin B2 (Riboflavin)

AOAC-PTM

Vitamin B6 (Pyridoxin)

Inositol

Vitamin C (L-Ascorbic Acid)

01/2023, page 8

Multi-Vitamin BVitamin testing solutions

How can you check for matrix effects in food sample ?
Different spiking standards are available from R-Biopharm AG to identify matrix effects.
Table 4: Spiking standard for VitaFast® test kits.
VitaFast®
Spiking standards

01/2023, page 9

Vitamins
Folic Acid Spiking standardVitamin B12 Spiking standardVitamin B7 (Biotin) Spiking standardPantothenic Acid Spiking standardVitamin testing solutions

Which testing approach is right for me?
Medium sized contract labs
Equipment
RIDA®ABSORBANCE 96
Test kits
VitaFast® test kits

"Microbiological assays are recognized by international
official institutions as the gold standard formany years. Microbiological methods allow thedetermination of both native and added vitamins.

microtiter plate wells. The test reagents are ready touse and standardized. The medium and standard only
have to be reconstituted with sterile water containedin the kit.

In the past we have used traditional microbiology
for vitamins. However, we had problems with media
production, contamination, maintaining of stock
cultures and procurement of raw materials. The
lab staff are enthusiastic about VitaFast® test kits,
where specific microorganisms are pre-coated in the

Depending on the food matrix different vitamins canbe determined from the same sample extract (seetable 5). Compared to traditional microbiologicalmethods, VitaFast® test kits significantly reduce
sample processing time and provide greater reliability,
higher productivity and better accuracy."

01/2023, page 10

Table 5: The sample preparation within the same food category and having the same color code are inprinciple combinable (white color = methods are not combinable).
Parameter

Art. No.

Folic Acid
Vitamin B12 (Cyanocobalamin)
Vitamin B7 (Biotin)
Vitamin B3 (Niacin)
Pantothenic Acid
Vitamin B1 (Thiamin)
Vitamin B2 (Riboflavin)
Vitamin B6 (Pyridoxin)

P1001
P1002
P1003
P1004
P1005
P1006
P1007
P1008

Total vitamin content ininfant food/baby food

Added vitamins in
capsules, pills,
vitamin mixes

Added vitamins in liquidsamples e.g. multivitamin juices, fitness drinks

Added vitaminsin cereals, flour

Vitamin testing solutions

Ingredients manufacturer
Equipment
RIDA®ABSORBANCE 96
Test kits• VitaFast® test kits
• RIDASCREEN®FAST Vitamin B12• RIDASCREEN®FAST Folic Acid

01/2023, page 11"We produce intermediate products containing
synthetic vitamins. We used to send our vitamin
samples (bars, powders, liquids, capsules and baked
goods) to an external lab, but this was very time
consuming and took at least 10 days. Investment in
HPLC and staff was too expensive and complicated.
We decided to use VitaFast® test kits. When startingwith VitaFast® we had to get used to sterile working
techniques like wearing gloves, sterile filtering andcleaning the workspace before starting. VitaFast® isreally user friendly and can be established quickly.

VitaFast® provides a high analytical performance.
Sometimes the impact of food processing might
have an impact on vitamins. For a quick processcontrol we use RIDASCREEN®FAST Vitamin B12 and
RIDASCREEN®FAST Folic Acid as they have the same
sample preparation and results are available within
two hours. With the ELISA we also check for vitaminhomogeneity in large food batches. The flexible
microtiter plate format is suited for small as well aslarge number of samples."Vitamin testing solutions

Govermental lab
Equipment
RIDA®ABSORBANCE 96
Test kits• VitaFast® test kits
• RIDASCREEN®FAST Vitamin B12"As a government lab our major role is to verify theconcentration of vitamins in the food with the productlabel. Sometimes at the end of the shelf life thevitamins have degraded. This is the reason why foodproducers add more vitamins than labelled. Dependingon the product we detect total or added vitamins.

01/2023, page 12

We stopped with traditional microbiology, because
we had quality problems and we needed to savelab space. We buy a whole range of VitaFast® test
kits. We do not carry out the sample preparation
to determine the total vitamin B12 content using
VitaFast® Vitamin B12 as it uses the toxic cyanide
and we want to protect our lab staff. Instead we use
RIDASCREEN®FAST Vitamin B12 to determine thetotal vitamin B12 content, the test kit has no toxic
sample preparation and results are available within
1 - 2 hours."

Vitamin testing solutions

Infant formula manufacturer
Equipment
HPLC
Test kits• EASI-EXTRACT® VITAMIN B12 (LGE)• EASI-EXTRACT® BIOTIN• VitaFast® test kits

"Babies are vulnerable consumers and as a result
infant formula is the most regulated food in the world.
It is therefore, vital for us to follow Official Methods
where possible. Both EASI-EXTRACT® VITAMIN B12(LGE) and EASI-EXTRACT® BIOTIN are referenced in
AOAC Final Action Official Methods therefore we felt
confident that these products could meet with thestrict performance criteria required and would provide
accurate and reliable results at the low levels we workat ensuring that we could confirm the claim levels of
our products.
Large format column to allow
filtrate to be stored in column
and mixed directly with gel.
No top frit in column.

01/2023, page 13

Top frit to keep the filtrate
separate from the gel.

The R-Biopharm LGE format column is unique andis the only column of this format on the market. LGE
columns are larger format column which allows the
sample to mix directly with the gel. We have observedslightly higher recoveries and better % RSD value."
For pantothenic acid we use VitaFast®
Pantothenic acid as it gives better results comparedto HPLC.

Figure 2: Immunoaffinity products

Vitamin testing solutions

Large contract lab
Equipment
HPLC
Test kits• EASI-EXTRACT® VITAMIN B12• EASI-EXTRACT® BIOTIN

01/2023, page 14"Although a high percentage of our vitamin testing is
conducted on infant milk formula/dairy industry weroutinely get a range of other samples to test for ourcustomers. We therefore require some versatility in
our methods to ensure we obtain optimum results
and as a result implemented immunoaffinity column
methods (EASI-EXTRACT® VITAMIN B12,
EASI-EXTRACT® FOLIC ACID and EASI-EXTRACT®
BIOTIN).

Extraction methods can easily be slightly modified if
required and technical support is always available from
R-Biopharm if we come across an unusual sample.
The range of application notes available is essential
to us and we routinely rely on these which helps us tomeet our quick turnaround times."

Vitamin testing solutions

Multi-national dairy company
Equipment
CHRONECT Symbiosis RIDA®CREST
Test kits
IMMUNOPREP® ONLINE VITAMIN B12"We previously only conducted small numbers ofvitamin analysis in-house using the R-Biopharmimmunoaffinity columns despite having our own
in-house laboratory and trained technical staff. The
majority of samples were sent to an external lab foranalysis.

01/2023, page 15

Costs for the external analysis were increasing
therefore we wanted to bring analysis in-house,focusing on vitamin B12 as over 70 % of our samples
were for this analyte. The aim was to reduce
overheads and to improve the speed of results. We
currently had to wait 4 - 6 working days. We routinely
sent 25 samples per week for analysis with a cost of>100 € per test.

It was important that the method implemented
had excellent performance and sensitivity as the
majority of samples tested were infant formulas.
Using the CHRONECT Symbiosis RIDA®CREST and
IMMUNOPREP® ONLINE VITAMIN B12 cartridges wehave been able to significantly reduce annual spend
on outsourcing and have been able to provide results
to our customers quicker. Overall, we have been
impressed that testing samples in-house is morecost effective, with the return on investment for the
CHRONECT Symbiosis RIDA®CREST being achieved
with a short time frame of around 18 months."

Vitamin testing solutions

Pharmaceutical company – validating themulti-vitamin column
Equipment
LC-MS/MS
Test kits
EASI-EXTRACT® MULTI-VIT B (LGE)"We have historically been using other methods forthe analysis of vitamins however we are re-assessing
our internal methods with the aim of improving ourworkflow. We therefore were looking at introducing a
range of methods for multi-analytes if possible.
We are using LC-MS/MS detection where possible
and one area that we were looking to improve was theanalysis of certain B-vitamins; namely vitamin B12and biotin. Our products are a range of multi-vitamins
which contain a range of minerals and antioxidants –many of which can cause issues during our current
extraction.

01/2023, page 16

We found out online that R-Biopharm had a rangeof immunoaffintiy columns, some of which wererecommended in AOAC Official Methods. This gave
us the confidence that the products were robust andcould meet with the strict recovery requirements that

we were looking for. After a consultation with theteam at R-Biopharm, we started to consider the
EASI-EXTRACT® MULTI-VIT B (LGE) columns insteadof the single vitamin products. EASI-EXTRACT®
MULTI-VIT B (LGE) is the only multi-vitamin column
on the market.
One of our initial challenges was to ensure we hada suitable sample preparation for our specific rangeof products. The technical team in Glasgow helped
us to develop a simple protocol which met with our
specifications. We are now in the final stages ofvalidating the method. We are looking forward to
finally implementing this method as we feel that it willbring many benefits to us whilst still ensuring that wereach our required performance specifications."

Appendix

VitaFast® – workflow

For this microbiological assay sterile working and a good pipetting technique is important. It is strongly
recommended to pipette samples and standards in triplicate. Depending on the sample preparation naturalor synthetic vitamins can be quantified. It is important to suspend the sample completely. Sample preparation
has to be done as outlined in the respective Instruction for Use. Important note: This workflow should describe
the general test procedure and the test principle. It does not replace reading of the respective IFU.

Test procedure

Test principle

1

Place the required number ofmicrowell strips in the frame

Microwells are coated with specificmicroorganisms2

Add 150 µL of assay medium

The addition of the assay medium
leads dissolves the microorganisms3

Add 150 µL of standard or sample

The growth of microorganism isdependent on the supply of therespective vitamin in the sample

4

Cover the strips with adhesive foil

It is important that the foil sticks
to the grid at every single well toprevent evaporation

Incubate the vitamin test according to the respective temperature and time (ref. IFU) in the dark
5
180˚

6

Press down the adhesive foil once
more. Place the microtiter plate upside
down on the table and dissolve themicroorganisms by shaking the plateon the surface of the desk

As the microorganisms havesettled to the bottom of the wellduring incubation, it is importantto mix the content of the well

Invert the plate to the regular
position and remove the adhesive
foil and remove remaining air
bubbles before measurement

The intensity of growth of the
microorganisms is correlated to theextracted vitamin and compared toa standard curve for quantitative
turbidimetrical measurement

01/2023, page 17

Read the plate in a microtiter plate photometer at 540 - 550 nm or 610 - 630 nm, use 4 parameter software
For evulation use the
RIDASOFT® Win.NET

7

low
conc.
conc.

VitaminMicroorganisms

high
conc.

The turbidity in each wellcorresponds to the growth ofthe microorganism