Amino acid Derivative Reactivity Assay(ADRA)
Standard Operating Procedure

Version 2.0
Pages:31
Annexes:0

Japanese Center for Validation of Alternative Methods (JaCVAM)
Skin Sensitization Validation Study

Issued by

Fujifilm corporation

Date:

Approved by

Validation Management Team

Date:

Distributed by

N/A

Revision History
Revision

Date:

Description of change:

Ver. 1.1

February
6, 20171) Revised NAL lot number on page 6.
2) Added a precautionary comment about pipette technique to section 4-2.
Preparation and Reaction of Reaction Solutions on page 13.
3) Revised the control criteria for the positive control reagent on page 23.
4) Added the range of average score and NAC depletion for additional test to
get an accurate predictive result in section 8-3. Data Acceptance for Amino
Acid Derivative Reactivity Assay on page 24.
5) Added instructions on how to handle instances in which there appears to be
no co-elution but depletion is less than -10% in section 8-4. Handling of
Co-elution on page 24.

Ver.1.2

April
2018

Ver.2.0

August 27, 1) Changed the concentration of the test chemical solution from 1 mM to 4
2021
mM and added the description about the 4 mM concentration.
2) Added a 0.5 mg/ml concentration of the test chemical solution for the
unknown molecular weight.
3) Added a description about fluorescence detection in the HPLC analysis.
4) Added squaric acid diethyl ester as another positive control.
5) Changed the range of the NAC/NAL depletion for the criteria of positive
control in section 8-1.
6) Added a description about the ADRA kit.
7) Updated references to the latest information.
8) Updated apparatus and reagents to the latest information in section 1.
9) Added caution about a sterilized 96 well plate in section 1-2.
10) Added caution about the quality of NAC and NAL in section 1-3.

16, 1) Added notes regarding solubility of test chemicals to page 9.

111) Added caution about the preparation of the reaction fixing solution insection 2-1-412) Added a note regarding filtration to remove the precipitate in section 5-1.

2

Table of Contents
Principle and Scope
References
1. Apparatus and Reagents
1-1. Apparatus
1-2. Consumables
1-3. Chemical and Special Materials
2. Pre-work
2-1. Preparation of each solution (For example)
2-2. Preparation of NAC and NAL stock solutions
2-3. Preparation of Standard Solution for Calibration Curve
2-4. Solubility Assessment of Test Chemicals
2-5. Preparation of HPLC system
3. Solution Preparation
3-1. Preparation of Test Chemical Solution
3-2. Preparation of Positive Control Solution
3-3. Thaw of each stock solution
4. Assay Procedure
4-1. Reference Control, Co-elution Control and Test chemical solution
4-2. Procedure of Preparation and Reaction of each reaction solution
4-3. Procedure after the Completion of Reaction
5. HPLC Analysis
5-1. HPLC Conditions
5-2. HPLC Sample Analysis Sequences
6. Data Analysis & Calculations
6-1. Calculation of Peak Area of NAC and NAL
6-2. Calculation of Concentration of NAC and NAL
6-3. Calculation of Peak Area of NAC and NAL
6-4. Calculation of Percent Depletion of NAC and NAL
7. Data Reporting
8. Acceptance Criteria
8-1. Acceptance Criteria for Amino acid Derivative Reactivity Assay Run
8-2. Acceptance Criteria for Each Test Chemical
8-3. Data Acceptance for Amino Acid Derivative Reactivity Assay
8-4. Handling of Co-elution
Example HPLC Analysis

3

Amino acid Derivative Reactivity Assay
PRINCIPLE AND SCOPE
The covalent binding of low molecular chemicals (haptens) to skin proteins is the molecular initiating
event for skin sensitisation. The Amino acid Derivative Reactivity Assay (ADRA) is used to evaluate thereactivity of a test chemical with N-(2-(1-Naphthyl)acetyl)-L-cysteine (NAC) and α-N-(2(1-Naphthyl)acetyl)-L-lysine (NAL) by combining the test chemical with a solution of NAC and NAL(abbreviated hereinafter as “NAC/NAL solution”) and then measuring the residual concentration of NAC
and NAL after a 24 hour incubation at 25ºC. NAC and NAL are chemically-synthesized compounds
containing a naphthalene group simulating cysteine and lysine residues in skin proteins. Residual
concentrations of NAC and NAL following the 24 hour incubation are determined by high performanceliquid chromatography (HPLC) with gradient elution and UV detection at 281 nm or fluorescence
detection with an excitation wavelength of 284 nm and an emission wavelength of 333 nm. Test
chemicals are prepared and analyzed in triplicate in batches of up to 17 chemicals, including controls. The
method is applicable to test chemicals that are soluble in acetonitrile or other non-reactive, water-miscible
solvents at 4 mM or 0.5 mg/ml (for test chemicals of unknown molecular weight) concentration.
REFERENCES
OECD. Test no. 442C APPENDIX II: In chemico skin sensitisation: Amino acid derivative reactivity
assay (ADRA). 2021. Retrieved from https://www.oecd-ilibrary.org/environment/test-no-442c-inchemico-skin-sensitisation_9789264229709-en
Fujita, M., et al., “Development of a prediction method for skin sensitization using novel cysteine andlysine derivatives.“, J. Pharm. Toxicol. Method. 2014; 70; 94-105.
Yamamoto, Y., et al., “A novel in chemico method to detect skin sensitizers in highly diluted reaction
conditions. “, J. Appl. Toxicol. 2015; 35: 1348-1360.
Yamamoto, Y., et al., “Expanding the applicability of the Amino acid Derivative Reactivity Assay(ADRA): Determining a weight concentration for preparation of test chemical solutions that yields a
predictive capacity identical to the conventional method using molar concentration and demonstrating thecapacity to detect sensitizers in liquid mixtures.”, J. Pharm. Toxicol. Method. 2019; 97: 67-79.
Imamura, M., et al., “Improving predictive capacity of the Amino acid Derivative Reactivity Assay test
method for skin sensitization potential with an optimal molar concentration of test chemical solution.”, J.
Appl. Toxicol. 2021; 41: 303-329.

4

1. APPARATUS AND REAGENTS
ADRA is best performed using disposable plastic labware, because NAC is susceptible to oxidization
by trace metal ions which may be found in glassware. Spatulae used for weighing test chemicals and thevarious parts of the HPLC are not subject to this restriction.
1-1. Apparatus (1)Apparatus

Example of use by Lead Laboratory

Analytical Balance

Capable of weighing up to 20 grams at an
accuracy of 0.1 mg readability

・Dispensing Pipettes capable of delivering 50–150 μl Verify accuracy at time of use
・12 Channel Dispensing Pipettes capable of delivering
50–150 μl
High Performance Liquid Chromatograph withlight-excluding auto-sampler for 96-well microwellplates capable of delivering a 0.3 ml/min flow rate

Shimadzu Prominence series

UV detector*1

Photodiode array detector (for example,
Shimadzu SPD-M20A) or absorbance detector(281 nm)

Fluorescence detector*2

Fluorescence detector (for example, Shimadzu
RF-20A)

pH meter with electrode and calibration

Capable of reading ±0.01 pH

HPLC Column

FUJIFILM Wako Pure Chemical Corporation,
Wakopak® Core C18 ADRA (2.6 μm, 3.0 × 150mm) [Catalog # 233-63991]
Alternative Column
The follow three columns have been confirmed
to yield results for five test chemicals that are
essentially the same as the FUJIFILM Wako
column.
OSAKA SODA, CAPCELL CORE C18 column(2.7 μm, 3.0 × 150 mm) [Cat. 51112]
ChromaNik, SunShell C18 (2.6 μm, 3.0 × 150mm) [Catalog # CB6371]
Waters, CORTECS C18 Column (2.7 μm, 3.0 ×
150 mm) [Catalog # 186007373]
Agilent, Poroshell 120 EC-C18 (2.7 μm, 3.0 ×
150 mm) [Catalog # 693975-302]

5

Guard Column and Guard Column Holder*3

Column: OSAKA SODA EXP GUARDCARTRIDGE CAPCELL CORE C18 S-2.7
2.1×5mm [Cat. 3643]
Holder: OSAKA SODA EXP DIRECTCONNECT HOLDER [Catalog # 3640]

Incubator

Capable of controlling at 25 +/ - 1ºC

1* If more than one wavelength is detectable, measurements should be made at both 281 nm and 291 nm
to confirm peak purities of NAC and NAL.
2
* Although a fluorescence detector is not always needed, it is useful when co-elution is observed in UV
detection.
*3
Guard columns need not be used for test chemicals that are not precipitated in the reaction solution.
1-1. Apparatus (2)Apparatus

Example of use by Lead Laboratory

Test Tube Mixer

SHIBATA Test Tube Mixer TTM-1

Plate Shaker

Heidolph Titramax 100 [Catalog # 544-11200-00]

Plate Centrifuge

KUBOTA PlateSpin

Recommended but non-essential consumables
Dispensing Pipettes capable of delivering 1000 –5000 μl

Verify accuracy at time of use

1-2. Consumables (1)Consumables

Recommended by Lead Laboratory

96-well Microwell Plate for HPLC analysis

Non-sterile polypropylene U bottom 96-well Microwell
Plate*1
Thermo (NUNC) U96 PP-0.5 ML NATURAL [Catalog# 267245]
Greiner MICROPLATE, 96 WELL, PP, U-BOTTOM,
NATURAL [Catalog # 650201]

96-well Microwell Plate seal for HPLC
analysis

Use the seal having sealing and solvent-resistantperformance*2
Shimadzu GLC TORAST™ 96well Seal E Type
[Catalog # 370-04030-01]*1

Sterilized plates should be avoided because changes in antioxidants contained in polypropylene due toheat, gamma rays, etc. may affect the reaction and/or be detected as contaminated peaks in HPLC.
*2
When using a plate seal other than the recommended one, ensure and verify that no part of the reaction
solution is volatilized and that the measured values are consistent before performing actual testing. Be
sure to leave no gaps between the plate seal and the plate when applying the plate seal.

6

1-2. Consumables (2)Consumables

Example of use (Used by Lead Laboratory)

Bottle with the quantity of approximately
500 mlWater sampling bottle (SANSEI MEDICAL) [Catalog #07-004-01]

Polypropylene centrifuge tube with thequantity equal to or more than 50 ml

100 ml: Watson Clean-vessel [Catalog # 536-150S]
50 ml: FALCON 50 ml PP Conical Tube [Catalog #352070]Measuring pipette for 10–100ml

100 ml measuring pipette: FALCON 100 ml Serological
Pipette [Catalog # 357600]
50 ml measuring pipette: FALCON 50 ml Serological
Pipette [Catalog # 356550]
25 ml measuring pipette: FALCON 25 ml Serological
Pipette [Catalog # 356525]

Polypropylene centrifuge tube with thequantity equal to or more than 10 ml

FALCON 15 ml Polypropylene Conical Tube [Catalog #352196]

Tube for cryopreservation

5 ml Tube*1: 5 ml Outer serum tube (SUMITOMOBAKELITE CO., LTD.) [Catalog # MS-4605X]
2 ml Tube*1: 2.0 ml Ring rock tube (BM Equipment Co.,
Ltd.) [Catalog # BM-20]
1.5 ml Tube*1: 1.7 ml Ring rock tube (BM Equipment
Co., Ltd.) [Catalog # BM-15]*1

A smaller or larger tube may be used per the quantity to be cryopreserved.

1-2. Consumables (3)Recommended but non-essential
consumables

Example of use by Lead Laboratory

Reservoir for operating 12 channel multipipette Corning 50 ml Reagent Reservoir [Catalog # 4870]
Approximately 1.5 ml tube and cap operable
by 12 channel multipipette(for dispensing test chemical solution)

Alphanumeric storage tube 1.4 ml (Thermo 4247JP)
SepraSeal (Thermo 4463)

Tool for removing multiple caps
simultaneously

Cap remover for SepraSeal (Thermo 4469)

1-3. Chemical and Special Materials
Chemical and Special Materials

Example of use by Lead Laboratory

ADRA kit

FUJIFILM Wako [Catalog # 296-80901]

Trifluoroacetic Acid (TFA)(MW=114.02) CASRN 76-05-1

FUJIFILM Wako [Catalog #204-02743]
98.0%

7

Sodium Phosphate, Monobasic, Anhydrous(NaH2PO4, MW=119.98)
CASRN 7558-80-7

FUJIFILM Wako [Catalog # 197-09705]

Sodium Phosphate, Dibasic, Anhydrous(Na2HPO4, MW=141.96)
CASRN 7558-79-4

FUJIFILM Wako [Catalog # 197-02865]

0.1M Sodium Hydroxide Solution
CASRN 1310-73-2

FUJIFILM Wako [Catalog # 196-02195]

Acetonitrile, HPLC Grade
CASRN 75-05-8

FUJIFILM Wako [Catalog # 015-08633]

Distilled waterDistilled water or equivalent may be used.

Acetone, special grade
CASRN 67-64-1

FUJIFILM Wako [Catalog # 016-00346]

Dimethyl sulfoxide (DMSO), special grade
CASRN 67-68-5

FUJIFILM Wako [Catalog # 043-07216]

Ethylenediamine-N,N,N',N'-tetraacetic acid,disodium salt, dihydrate (EDTA･2Na･2H2O)
CASRN 6381-92-6

Dojindo Molecular Technologies, Inc. [Catalog#343-01861

NAC (refrigeration storage) *1
MW=289.35

FUJIFILM Wako [Catalog # 294-83001]

NAL (refrigeration storage) *1
MW=314.38

FUJIFILM Wako [Catalog # 290-83101]

Phenyl acetaldehyde (Positive control)
MW=120.15 CASRN 122-78-1

SIGMA [Catalog # 107395]
Alfa Aesar [Catalog # A14263]
Note: Store phenyl acetaldehyde under thefollowing conditions.
(1) Store at a temperature of 2–8ºC. Do not store at
below 0ºC.
(2) Do not expose to UV light during storing or
handling.
(3) Handle as little as possible, keep stored under
the above conditions.

Squaric acid diethyl ester (Positive control)
MW=170.16 CASRN 5231-87-8

Tokyo Chemical Industry [Catalog # D3253]
SIGMA [Catalog # 107395](1) Store away from high temperature or humidity.

*1

The following qualities are required for NAC and NAL
1) Purity: Both NAC and NAL should exhibit at least 98% purity.
2) Stability: Using NAC and NAL stock solution, prepare a reference control that is free of any test
chemical and quantify the residual levels of both NAC and NAL immediately after preparation (0hours) and after a 24-hour incubation. Residual levels of NAC and NAL should be at a minimum of
90% in either case. The residual level of NAC is calculated as a percentage of the sum of NAC and8the residual level of NAC dimers.
3) Reactivity: NAC and NAL are to be evaluated for reactivity with the ten proficiency substances
described in APPENDIX II of OECD TG442C and should satisfy the requirement given therein.

2. PRE-WORK
Prepare the reagents according to the attached instructions when using the ADRA kit for the test.
2-1. Preparation of each solution (Typical)
2-1-1. 0.1 mM EDTA solution
1) Weigh 37.2 mg of EDTA･2Na･2H2O into a 15 ml Conical Tube and use a 25 ml measuring pipette to
add 10 ml of distilled water to dissolve it. (10 mM EDTA solution)2) Use a 50 ml measuring pipette to add 49.5 ml of distilled water to a 100 ml tube and add 0.5 ml of the
10 mM EDTA solution described above to dilute it 100 fold. (0.1 mM EDTA solution)
2-1-2. 100 mM Phosphate buffer (pH 8.0)
To prepare an approximately 300 ml quantity
1) Weigh 0.6 g of Sodium Phosphate Dibasic Anhydrous into a 100 ml tube and use a 50 ml measuring
pipette to add 50 ml of distilled water to dissolve 0.6 g of Sodium Phosphate Dibasic Anhydrous indistilled water.*1
2) Use a 50 ml (or 100 ml) pipette to add 300 ml of distilled water.
3) Weigh 4.26 g of Sodium Phosphate Monobasic Anhydrous and dissolve it in distilled water 2).*1
4) Use a 25 ml measuring pipette to add 16 ml of Sodium Phosphate Dibasic solution 1) in Sodium
Phosphate Monobasic solution 3).
5) Use a 25 ml measuring pipette to remove 17 ml from solution 4) and to add 1 ml of 0.1 mM EDTA
solution to solution 4). The concentrations of EDTA in this solution and final concentration of reaction
solution are 0.33 μM and 0.25 μM, respectively.
6) Measure pH of solution 5) using a calibrated pH meter and verify that the pH is between 7.9 and 8.1.
If the pH is out of range, prepare a new batch of phosphate buffer.
7) Use the NAC stock solution within a week or cryopreserve the unused quantity. Repackage it into 2
ml tubes to cryopreserve for Co-elution Control at a temperature below −75°C.
2-1-3. 100 mM Phosphate buffer (pH 10.2)
To prepare an approximately 300 ml quantity
1) Use a 50 ml measuring pipette to add 286 ml of distilled water to a 500 ml bottle.
2) Weigh 4.26 g of Sodium Phosphate Monobasic Anhydrous and dissolve it in the distilled water 1)*1.
3) Use a 25 ml measuring pipette to add 14 ml of 0.1M NaOH solution to the solution 2).
4) Measure the pH of solution 3) using a calibrated pH meter and verify that it is between 10.1 and 10.3.
If the pH is out of range, prepare a new batch of phosphate buffer.
5) Use the NAL stock solution within a week or cryopreserve the unused quantity. Repackage it into 2
ml tube to cryopreserve for Co-elution Control at a temperature below −75°C.
92-1-4. Reaction fixing solution (2.5% (v/v) TFA aqueous solution)
Add 2.5 ml of TFA to 100 ml of distilled water.*2
2-1-5. HPLC mobile phase A: 0.1% (v/v) TFA aqueous solution
Add 1.0 ml of TFA to 1000 ml of distilled water.
2-1-6. HPLC mobile phase B: 0.1% (v/v) TFA Acetonitrile solution
Add 1.0 ml of TFA to 1000 ml of HPLC grade Acetonitrile.
*1

When dissolving phosphate salts, close the bottle to mix them. Do not use glass labware or otherwashable equipment. Verify that salts are completely dissolved after mixing.
*2
Since the peak shape of NAC may be bimodal when the TFA concentration in the reaction solution
during HPLC measurement is low, be sure to measure the reagents accurately.
2-2. Preparation of NAC and NAL stock solutions
2-2-1. Preparation of NAC stock solution:
The NAC (molecular weight of 289.35) stock solution used in each assay is to all be from a single batch.
We recommend storing NAC stock solution in quantities sufficient for an assay. A typical preparation as
performed at the Lead Laboratory is described below.*1
1) Weigh 11.6 ± 0.1 mg of NAC in a 50 ml tube, add 20 ml of 100 mM phosphate buffer (pH 8.0) to thetube using a 25 ml measuring pipette, and mix it gently using a test tube mixer to dissolve the NAC.
(2 mM NAC stock solution).*22) Use a 50 ml measuring pipette to add 149.5 ml of this buffer to a 500 ml bottle. Next, add 0.5 ml of 2mM NAC stock solution to this solution and mix gently but thoroughly by inversion. (6.667 μM NAC
stock solution).
3) Cryopreserve 3 ml quantities of stock solution in cryopreservable 5 ml tubes at a temperature below−75°C. Store for no more than six months before use.*3
2-2-2. Preparation of NAL stock solution
The NAL (molecular weight of 314.38) stock solution used in each assay is to all be from a single
batch. We recommend storing NAL stock solution in quantities sufficient for an assay. A typicalpreparation as performed at the Lead Laboratory is described below.*1
1) Weigh 12.6 ± 0.1 mg of NAL in a 50 ml tube, and add 20 ml of 100 mM phosphate buffer (pH 10.2)
to the tube using a 25 ml measuring pipette, and mix it gently using a test tube mixer to dissolve theNAL. (2 mM NAL stock solution).*22) Use a 50 ml measuring pipette to add 149.5 ml of this buffer to a 500 ml bottle. Next, add 0.5 ml of
2 mM NAL stock solution to this solution and mix it gently but thoroughly by inversion. (6.667 μM
NAL stock solution)
3) Cryopreserve 3 ml quantities of stock solution in 5 ml tubes at a temperature below −75°C. Store forno more than six months before use.*3*1
These quantities are typical examples only and can be adjusted as necessary.
10

*2

The remaining 2 mM stock solution can be cryopreserved at a temperature below −75°C for later use.
Typically, cryopreserve 1 ml of stock solution in a 1.5 ml tube at a temperature below −75°C.
*3
Stability testing of NAC stock solution at temperatures of 25ºC, 4ºC, -20ºC, and -80ºC has shown thatit remains stable and free of auto-oxidation only at -80ºC. We recommend that the NAL stock solution
also be stored at a temperature below −75°C, although NAL is not susceptible to oxidization in thesame manner as NAC.
Note: Prior to preparing new batches of NAC/NAL stock solution, verify the stability of the NAC/NAL
stock solution using the following procedure.
1) Add 150 μl of 6.667 μM NAC/NAL stock solution to three wells in each of two 96-well Microwell
Plates.
2) Add 50 μl of acetonitrile to each of these six wells 1).
3) Attach a plate-seal to one of the two 96-well Microwell Plates and spin down the plate using acentrifuge, the incubate for 24 hours at 25ºC. After incubation, add 50μl of reaction fixing solution toeach well, attach a new plate-seal, then mix and spin down the plate. Analyze NAC/NAL using the
HPLC system.
4) Add 50 μl of reaction fixing solution to the other 96-well Microwell Plate, attach a plate-seal to it,then mix and spin down the plate. Analyze NAC/NAL using the HPLC system.
If the residual concentration of NAC/NAL falls below 90% after either 0 hour or 24 hours, the NAC and
NAL are not stable, and so new batches of NAC/NAL solutions must be prepared.
2-3. Preparation of Standard Solution for Calibration Curve
The calibration curve for the acceptance criteria is obtained from a six-dose standard solution. The initialstandard solution, Std1 at 5.0 μM, is cryopreserved in small quantities and used to prepare dilution series.
A typical procedure for preparation of the standard solution is described below.*1
Procedure for preparation of 20 ml solution and 40–66 tubes
1) Use a 25 ml pipette to add 15 ml of 6.667 μM NAC/NAL stock solution to a 50 ml tube. Then use amicropipette to add 1 ml of distilled water, 100μl of TFA, and 3.9 ml of acetonitrile.
2) Cryopreserve 300–500 μl quantities of the standard solution in 1.5 ml tubes at a temperature below−75°C.
Also, solvent for preparing dilution series of the standard solution may be cryopreserved in 1000–1200
μl quantities, as described below.
1) Use a 25 ml pipette to add 15 ml of 100 mM phosphate buffer (pH 8.0 for NAC, pH 10.2 for NAL), 1
ml of distilled water, 100 μl of TFA, and 3.9 ml of acetonitrile to a 50 ml tube.
2) Typically, cryopreserve approximately 1 ml of solvent in a 1.5 ml tube at a temperature below −75°C.
*1These quantities are typical examples only and can be adjusted as necessary.
2-4. Solubility Assessment of Test Chemicals
The solubility of each test chemical in a suitable solvent must be assessed before performing the actual
assay. A suitable solvent will dissolve the test chemical completely with no apparent turbidity or
11precipitate during visual inspection. The concentration of test chemical solution used in the assay is 4
mM, but more than 10 mg is recommended to be weighed because of the precise weight of the test
chemical. For this reason, the initial concentration of the test chemical solution is set to 20 mM, and thefinal 4 mM test chemical solution is prepared by 5-fold dilution of the 20 mM solution. For a solvent of
DMSO, the test chemical solution is set to 80 mM. This solution should then be diluted 20-fold withacetonitrile to prepare 4 mM test chemical solution. For test chemicals of unknown molecular weight,
the initial concentration of the test chemical solution is set to 10 mg/ml, and the final 0.5 mg/ml test
chemical solution is prepared by a 20-fold dilution of the 10 mg/ml solution. The test chemical isdissolved using a test tube mixer and an additional 5–10 minutes of sonication if needed due to low
solubility. Because the quantity of test chemical is small relative to the quantity of solvent, solubility is
to be carefully verified macroscopically.
The four solvents below are all suitable for used in ADRA, and the priority for the selection of theappropriate solvent is as follows.
(1) Distilled water(2) Acetonitrile(3) Acetone(4) DMSO
Note 1: When the 80 mM or 10 mg/ml solution is prepared using DMSO (4), use acetonitrile to prepare
the 20-fold dilution down to a 4 mM solution.
Note 2: If none of these solvents are suitable for a given test chemical in a 20 mM or 10 mg/ml solution,assess the solubility of the test chemical in 4 mM or 0.5 mg/ml solution using solvents (1)–(3). Do not
use DMSO to prepare a 4 mM or 0.5 mg/ml solution. DMSO must always be diluted 20 fold withacetonitrile to prepare a 4 mM or 0.5 mg/ml solution.
Note 3: Water is not suitable as a solvent for anhydrides due to hydrolysis.
A typical procedure is described below.
1. Use the following formula to calculate the quantity of test chemical needed to prepare approximately
5 ml of a 20 mM test chemical solution, 25 ml of a 4 mM test chemical solution or 5 ml of an 80 mM
test chemical solution. If no purity information is available, assume 100% purity.
Preparation of the 20 mM solution (5 ml) or 4 mM solution (100 ml)
MW ×

100
20 mM
×
× 5 mlPurity (%)
1000= MW ×

or

MW ×

100
4 mM
×
× 25mlPurity (%)
1000

10= Required quantity (mg)
Purity (%)

Preparation of the 80 mM solution (5 ml)
MW ×

100
80 mM
40
= Required quantity (mg)×× 5 ml = MW ×
Purity (%)
1000
Purity (%)

12

2. Weigh the required quantity of test chemical directly into a centrifuge tube or other suitable labware
and record the actual weight. For test chemicals of unknown molecular weight, weigh more than 10 mgof test chemical solution directly into a centrifuge tube or another suitable labware and record theactual weight.
3. Calculate the required quantity of solvent using the following formula, and assess the solubility for theappropriate solvent.
Preparation of the 20 mM solution
Test chemical (mg) ×

1
MW= Test chemical (mg) ×

Purity (%)×
MW×

Purity (%)
1
×× 1000 ml
100
20 mmol= Volume of solvent (ml)0.5

Preparation of the 4 mM solution
Test chemical (mg) ×

1
MW= Test chemical (mg) ×

Purity (%)×
MW×

Purity (%)×
100

1
4 mmol× 1000 ml

2.5= Volume of solvent (ml)0.125

= Volume of solvent (ml)Preparation of the 80 mM solution= Test chemical (mg) ×

Purity (%)×
MWPreparation of the 10 mg/ml solution
Test chemical (mg) ×

Purity (%)×
100= Test chemical (mg) ×

1
10 mg

Purity (%)= Volume of solvent (ml)1000

Preparation of the 0.5 mg/ml solution
Test chemical (mg) ×

Purity (%)×
100= Test chemical (mg) ×1
0.5 mgPurity (%)= Volume of solvent (ml)50Note 1: Even if the required quantity is calculated to be less than 10 mg, weigh more than 10 mg of test
chemical for precise weight.
Note 2: If the test test chemical solution is in aqueous form, calculate necessary volume of the aqueous
solution and quantity of water for dilution using the following formula. If specific gravity of test
chemical solution is not known, assume it as 1.0.

13

Preparation of the 20 mM solution (5 ml)
100
1
20 mM × 5 ml
×× MW ×× 1000 (μl)
Specific gravity (g/ml) × 1000 ml
Concentration (w/w%)
1000
=

10
MW
= Volume of test chemical (μl)×
Concentration (w/w%)
Specific gravity (g/ml)

5000 μl － Volume of test chemical (μｌ) = Solvent volume (μl)

Preparation of the 4 mM solution (25 ml)
100
1
4 mM × 25 ml
×× MW ×× 1000 (μl)
Specific gravity (g/ml) × 1000 ml
Concentration (w/w%)
1000
=

10
MW
= Volume of test chemical (μl)×
Concentration (w/w%)
Specific gravity (g/ml)

25 ml －

Volume of test chemical (μｌ)= Solvent volume (ml)1000

Preparation of the 10 mg/ml solution (1 ml)
100
10 mg/ml × 1 ml
×× 1000 (μl)
Specific gravity (g/ml) × 1000 (mg)Concentration (w/w%)
=

1000
1
= Volume of test chemical (μl)×
Concentration (w/w%)
Specific gravity (g/ml)

1000 μl － Volume of test chemical (μｌ) = Solvent volume (μl)

Preparation of the 0.5 mg/ml solution (20 ml)
0.5 mg/ml × 20 ml
100
×× 1000 (μl)
Specific gravity (g/ml) × 1000 (mg)Concentration (w/w%)

=

1000
1
= Volume of test chemical (μl)×
Concentration (w/w%)
Specific gravity (g/ml)

1000 μl － Volume of test chemical (μｌ) = Solvent volume (μl)

2-5. Preparation of HPLC System
Prepare an HPLC system with a 281-nm UV detector and/or a 284-nm/333 nm (Ex/Em) FL detector.
Prepare mobile phases A and B as described in section “2-1-5. HPLC mobile phase A: 0.1% (v/v) TFA
aqueous solution” and in section “2-1-6. HPLC mobile phase B: 0.1% (v/v) TFA Acetonitrile solution”.
1. Column Equilibration: Install the HPLC column (see section “1.1 Apparatus (1)”) and equilibrate thecolumn for at least 30 minutes at 40ºC with 50% A, 50% B before use. Then, condition the column byrunning the gradient at least twice before using the column.
2. System Shutdown: Following analysis, maintain a low flow (typically 0.05 ml/min) of 50% A and
50% B through the system and decrease column temperature to approximately 25ºC.
3. If the system is to be idle for more than a week, fill the column with acetonitrile (without
14

Trifluoroacetic acid) and remove the column from the HPLC system. Cap tightly and purge acidcontaining mobile phases from the system using acetonitrile.

3. SOLUTION PREPARATION
3-1. Preparation of Test Chemical Solution
Solubility of the test chemical in the appropriate solvent is evaluated in section “2-4. Solubility
Assessment of Test Chemicals”. 20 mM, 4 mM, 80 mM, 10 mg/ml, or 0.5 mg/ml solutions of test
chemicals in the appropriate solvents are prepared immediately before use.
1. When ready to perform the assay, calculate and weigh the appropriate quantity of test chemical
needed to prepare a 20 mM, 4 mM, 80 mM, 10 mg/ml, or 0.5 mg/ml solution directly in a 15 ml orother appropriate tube. Then calculate the required quantity of solvent needed based on the actual
weight of the test chemical. In the above operation, use the formula described in section “2-4.
Solubility Assessment of Test Chemicals” to calculate the required quantities of the test chemical andof the solvent.
2. Add the volume of solvent calculated and mix in a centrifuge tube, using a test tube mixer to dissolve
the test chemical. Slight sonication (less than 5 minutes) may be used if needed. If the test chemical is
not completely dissolved, do not proceed with that specific test chemical in the selected solvent.
Re-evaluate with another solvent (see section “2. PRE-WORK”) to find a suitable vehicle.
3. Dilute 20 mM, 80 mM, or 10 mg/ml of test chemical solution to the final 4 mM or 0.5mg/ml solution
with each appropriate solvent in the 1.4 ml Test chemical solution Storage Tube or other appropriate
tube (For example, add 200 μl of 20 mM test chemical solution to 800 μl of appropriate solvent, add
50 μl of 80 mM or 10 mg/ml test chemical solution to 950 μl of appropriate solvent).*1
4. Record and report the final solvent choice for each chemical if the final solvent is different from thesolvent selected in section “2-4. Solubility Assessment of Test Chemicals”.
*1
96-well Deepwell Plate may be used instead of 1.4 ml Test chemical solution Storage Tube.
3-2. Preparation of Positive Control Solution
Phenyl acetaldehyde (Molecular weight=120.15) or squaric acid diethyl ester (molecular weight =170.16) dissolved in acetonitrile is used as Positive Control and should be contained in each run of assay.
A typical preparation procedure for the Positive Control solution is described below.
1. Calculate a weight of Positive Control to be needed to prepare approximately 5 ml of 20 mM Positive
Control solution from following formula. The next formula shows the case that the purity of Phenyl
acetaldehyde is 90%.
MW ×

10= 120.15 ×
Purity(%)

10
90= 13.35 (mg) = Phenyl acetaldehyde (mg)

2. Weigh the phenyl acetaldehyde directly in a 10- or 15 ml centrifuge tube, and record the actual
weight.
3. Calculate necessary quantity of solvent from following formula based on the actual weight.
Phenyl acetaldehyde (mg) ×

Purity (%)
× 0.5
MW
15

= 13.35 ×90
× 0.5 = 5.0 (ml) = Solvent volume (ml)120.15

4. Dissolve the positive control with acetonitrile in the quantities calculated above (20 mM).
5. Dilute 20 mM of positive control solution 5-fold with acetonitrile in the 1.4 ml Test chemical solution
Storage Tube or other appropriate tube. (final concentration or 5 mM)*1*1

96-well Deepwell Plate may be used instead of 1.4 ml Test chemical solution Storage Tube.

3-3. Thaw of each stock solution
Thaw frozen Phosphate buffer (pH 8.0 and 10.2) and NAC/NAL stock solution at room temperaturewhen using frozen stock solutions. The preparation of each stock solution is described in sections “2-1-2.
100 mM Phosphate buffer (pH 8.0)”, “2-1-3. 100 mM Phosphate buffer (pH 10.2)”, and “2-2. Preparationof NAC/NAL stock solutions”.
4. Assay Procedure
4-1. Reference Control, Co-elution Control and Test Chemical Solution
Test chemical solutions are prepared in triplicate for NAC/NAL. Each assay (NAC/NAL) may be
prepared and performed concurrently (NAC/NAL should not be incubated and analyzed on separate days).
The three Reference Control A, six Reference Control C and one Co-elution Control (the solution after
addition of buffer solution rather than NAC/NAL) should be prepared. Moreover, the solution (Co-elution
Control, N=1) for each test chemical after addition of buffer solution rather than NAC/NAL solution
should be prepared. The detail of each solution describes below.
Test chemicalsolution

Reaction solution of NAC/NAL with test chemical. Evaluate reactivity of
NAC/NAL with the test chemical at a ratio of 1:200 for the NAC/NAL with thetest chemical.

Reference Control A

Control for verifying validity of the HPLC system. Reference Control A is used toverify concentration of NAC/NAL from each calibration curve after addition ofacetonitrile rather than test chemical.

Reference Control B

Control for verifying stability of reaction solution under analysis. Reference
Control B is used to verify variability (CV) of each three NAC/NAL peak areas in
the solution after addition of acetonitrile rather than test chemical at the start ofanalysis and at the end of analysis.

Reference Control C

Control for calculating NAC/NAL depletion of each test chemical solution. To
calculate depletion of NAC/NAL, measure three Reference Controls C after
addition of solvent instead of test chemical. Prepare reference Control C for all
solvents used to dissolve the test chemicals.

Co-elution Control

Control for verifying whether or not test chemicals co-elute with NAC/NAL. The
Co-elution Control is used to verify absorbance at 281 nm and/or fluoresces at 284
nm/333 nm (Ex/Em) and whether retention time of test chemical is equal to that of
NAC/NAL.

16

4-2. Preparation and Reaction of Reaction Solutions
Use 12-channel pipettes with 96-well plates. Proper attention is required when using pipettes: pre-rinse
at least five times to ensure precise pick up and take care to avoid dripping from the tip when dispensing.
1. Assemble the following previously prepared reagents, solvents, and solutions:a. 6.667 μM of NAC/NAL stock solution,b. Appropriate buffer solution (pH 8.0 for NAC, pH 10.2 for NAL),
c. Test chemical solution (or solvent for Reference Controls)
2. Use a 96-well Microwell Plate and 12-channel pipette to prepare test chemical solutions by adding thereagents per the following table. Refer to the section on “Typical Test Chemical Solution Positions in
96-well Microwell Plate” for typical positions in the plate. Inspect test chemical solution after adding
test chemical and record observations.
Ratio of NAC to test chemical = 1:200
5 μM NAC, 1 mM test chemical

Ratio of NAL to test chemical = 1: 200
5 μM NAL, 1 mM test chemical

・150 μl NAC solution
・50 μl Test chemical solution

・150 μl NAL solution
・50 μl Test chemical solution

Co-elution Control and Reference Control A, B and C are prepared according to the following tables.
Co-elution Control
For NACFor NAL

・150 μl Phosphate buffer (pH 8.0)
・50 μl Test chemical solution

・150 μl Phosphate buffer (pH 10.2)
・50 μl Test chemical solution

Reference Control A and B
For NACFor NAL

・150 μl NAC solution
・50 μl Acetonitrile

・150 μl NAL solution
・50 μl Acetonitrile

Reference Control C
For NACFor NAL

・150 μl NAC solution
・50 μl Acetonitrile and solvent used for
dissolution of test chemical *1*1・150 μl NAL solution
・50 μl Acetonitrile and solvent used for
dissolution of test chemical *1

If the test chemicals are dissolved in acetonitrile, only acetonitrile need be prepared.

3. Seal the 96-well Microwell Plate completely with a plate seal and shake it using a Plate Shaker.
Centrifuge the plate using a Plate Centrifuge and incubate the plate in the Incubator (dark) at 25ºC
(plus or minus 1’C) for 24 hours (plus or minus 1 hour). Record the time and temperature at the startand end of incubation.
4-3. Procedure after the Completion of Reaction
1. After 24 hours of incubation, remove the plate seal and add 50 μl of Reaction Fixing Solution to eachtest chemical solution. Inspect the reacted test chemical solution and record any observations after
addition of the reaction fixing solution. If precipitation is observed, centrifuge the 96-well Microwell
Plate at low speed (100–400g) to force the precipitate to the bottom of the well as a precaution. If more
17

than 100 μl of supernatant of test chemical solution can be removed using a pipette, the supernatant may
be analyzed after being transferred to another plate.
2. Prepare the standard solution
Prepare NAC/NAL standard solutions by serial dilution from 5.0–0.156 μM(1) Prepare each solution ahead of time after thawing frozen stock solution at room temperature.
[1] 5.0 μM stock solution “Std1”[2] “solvent for preparing dilution series” (for NAC/NAL)(2) Prepare the following concentration of the standard solution by diluting 150 μl of stock solution“Std1” with the same quantity of dilution solvent per to “Preparation procedure for dilution series”
below. Add dilution solvent to a well as “Std7”.

NAC/NAL
(μM)

Std 1

Std 2

Std 3

Std 4

Std 5

Std 6

Std 7
(Dilution solvent)

5.0

2.5

1.25

0.625

0.313

0.156

0

“Preparation procedure of dilution series”(1) Add 150 μl of dilution buffer to wells from well Std2 to well Std7 shown in “Typical Test Chemical
Solution Positions in 96-well Microwell Plate”(2) Add 150μl of Std1 to well Std1 and well Std2. Mix with minimal air entrapment.
(3) Transfer 150 μl from well Std2 to well Std3. Mix with minimal air entrapment.
(4) Continue in a similar manner from well Std4 to well Std6.
3. Prepare dilution series of NAC/NAL standard solution in the wells from Std1 to Std7 shown in the next
section on “Typical Test Chemical Solution Positions in 96-well Microwell Plate”, and reseal the platetightly with a new plate seal. For preparation of dilution series, see section “4-3. 2. Prepare thestandard solution”. Mix the plate with a Plate Shaker and spin it down by centrifuge.
Note: Add the Reaction Fixing Solution within 30 minutes of the end of incubation.
Typical Test Chemical Solution Positions in 96-well Microwell Plate
Between 1 and 17 test chemicals can be tested in a single test run using two 96-well plates, based on
consideration of the time needed for preparation and testing as well as the burden on laboratory
technicians and the capacity of the HPLC apparatus. If more than one solvent are used a single test run,additional Reference Controls for each solvent need to be prepared and the number of test chemicals willneed to be reduced. For example, if three kinds of solvent are used, the maximum number of test
chemicals is 15. The number of test chemicals and Typical Test chemical solution position in 96-well
Microwell Plate are shown below, but the positions may be adjusted per the number of test chemicalsolutions.

18

(1) For five test chemicals with acetonitrile as solvent

N
A
C
N
A
L

1

2

3

4

5

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

RC-A

C

Test
chemicalsolution
No.1

D

CC-1

CC-2

CC-3

CC-4

CC-5

－

E

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

RC-A

G

Test
chemicalsolution
No.1

H

CC-1

CC-2

CC-3

CC-4

CC-5

－

A
B

F

6

7
RC-B
－
RC-B
－

8
RC-B
－
RC-B
－

9
RC-C
－
RC-C

10
PC
－
PC

11

12

STD 1

STD 5

STD 2

STD 6

STD 3

STD 7

STD 4

－

STD 1

STD 5

STD 2

STD 6

STD 3

STD 7

－

－

STD 4

－

RC: Reference Control, CC: Co-elution Control, PC: Positive Control(2) For 10 test chemicals with acetonitrile and water as solvent
First plate

N
A
C
N
A
L

1

2

3

4

5

6

7

8

9

10

11

12

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

Test
chemicalsolution
No.6

Test
chemicalsolution
No.7

Test
chemicalsolution
No.8

Test
chemicalsolution
No.9

Test
chemicalsolution
No.10

－

－

C

Test
chemicalsolution
No.1

D

CC-1

CC-2

CC-3

CC-4

CC-5

CC-6

CC-7

CC-8

CC-9

CC-10

－

－

E

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

Test
chemicalsolution
No.6

Test
chemicalsolution
No.7

Test
chemicalsolution
No.8

Test
chemicalsolution
No.9

Test
chemicalsolution
No.10

－

－

G

Test
chemicalsolution
No.1

H

CC-1

CC-2

CC-3

CC-4

CC-5

CC-6

CC-7

CC-8

CC-9

CC-10

－

－

2

3

4

5

6

7

8

9

10

11

12

RC-C

RC-C

STD 1

STD 5

STD 2

STD 6

STD 3

STD 7

STD 4

－

STD 1

STD 5

STD 2

STD 6

STD 3

STD 7

STD 4

－

A
B

F

Second plate
1
N
A
C
N
A
L

A
B

RC-A

RC-B

RC-B

C
D

－

－

－

E
F

RC-A

RC-B

RC-B

G
H

－

－

－

(Water) (ACN)
－

－

RC-C

RC-C

(Water) (ACN)
－

－

PC
－
PC
－

－

－

－

－

－

－

－

－

－

－

－

－

－

－

－

－

RC: Reference Control, CC: Co-elution Control, PC: Positive Control, ACN: Acetonitrile

19

(3) For 17 test chemicals and acetonitrile as solvent
First plate

N
A
C
N
A
L

1

2

3

4

5

6

7

8

9

10

11

12

C

Test
chemicalsolution
No.1

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

Test
chemicalsolution
No.6

Test
chemicalsolution
No.7

Test
chemicalsolution
No.8

Test
chemicalsolution
No.9

Test
chemicalsolution
No.10

Test
chemicalsolution
No.11

Test
chemicalsolution
No.12

D

CC-1

CC-2

CC-3

CC-4

CC-5

CC-6

CC-7

CC-8

CC-9

CC-10

CC-9

CC-10

E
G

Test
chemicalsolution
No.1

Test
chemicalsolution
No.2

Test
chemicalsolution
No.3

Test
chemicalsolution
No.4

Test
chemicalsolution
No.5

Test
chemicalsolution
No.6

Test
chemicalsolution
No.7

Test
chemicalsolution
No.8

Test
chemicalsolution
No.9

Test
chemicalsolution
No.10

Test
chemicalsolution
No.11

Test
chemicalsolution
No.12

H

CC-1

CC-2

CC-3

CC-4

CC-5

CC-6

CC-7

CC-8

CC-9

CC-10

CC-11

CC-12

1

2

3

4

5

6

7

8

9

10

11

12

Test
chemicalsolution
No.14

Test
chemicalsolution
No.15

Test
chemicalsolution
No.16

Test
chemicalsolution
No.17

STD 1

STD 5

RC-A

STD 2

STD 6

C

Test
chemicalsolution
No.13

STD 3

STD 7

D

CC-3

CC-4

CC-5

CC-6

CC-7

－

STD 4

－

E

Test
chemicalsolution
No.14

Test
chemicalsolution
No.15

Test
chemicalsolution
No.16

Test
chemicalsolution
No.17

STD 1

STD 5

STD 2

STD 6

G

Test
chemicalsolution
No.13

STD 3

STD 7

H

CC-13

CC-14

CC-15

CC-16

CC-17

STD 4

－

A
B

F

Second plate

N
A
C
N
A
L

A
B

F

RC-A

RC-B
－
RC-B

RC-B
－
RC-B

RC-C
－
RC-C
－

PC
－
PC
－

RC: Reference Control, CC: Co-elution Control, PC: Positive Control, ACN: Acetonitrile

5. HPLC Analysis
1. Install the appropriate column in the HPLC system, prime and equilibrate the entire system with the
Mobile Phase A and Mobile Phase B at column temperature of 40°C. The HPLC analysis is performed
using a flow of 0.3 ml/min and a linear gradient from 30% to 55% acetonitrile for NAC and from 25%
to 45% acetonitrile for NAL within 10 minutes, followed by a rapid increase to 100% acetonitrile toremove other materials. Refer “5-1. HPLC Conditions” for details on the gradient.
2. Inject equal quantities of each standard solution, test chemical solution, and control solution. The
injection quantity varies according to the system used but typically is from 10–20 μl. Systems require
smaller injection quantities, because injection of 20 μl leads to unacceptably broad peaks. Absorbanceis monitored at 281 nm. If using a Photodiode Array Detector, absorbance at 291 nm should also berecorded. When co-elution of the test chemical with NAC or NAL is observed, fluorescence detection(284 nm for excitation and 333 nm for emission) is available.
3. Increase the mobile phase B to 100% in order to remove the other compounds from the column after
gradient, and re-equilibrate the column under initial conditions for at least 6.5 minutes. These processof acetonitrile 100% and re-equilibration for 6.5 minutes are described the following table “5-1.
20

HPLC Conditions”.
Note 1: If NAC and NAL peaks are saturated due to the performance of the fluorescence detector, eachsolution may be diluted 2–10 fold before measurement. However, the solution composition(concentration of buffer and TFA) after dilution should be the same as that before dilution sincechanges of the pH in the solution may affect measurement results.
Note 2: The 6.5 minute re-equilibration time was determined using a Shimadzu Prominence HPLC system.
Other systems may require more or less re-equilibration time due to system mixing quantity. Shorter
equilibration times will be acceptable if peak retention times are stable.
5-1. HPLC Conditions
Column

FUJIFILM Wako, Wakopak® Core C18 ADRA (2.6μm, 3.0 × 150 mm) [Catalog # 233-63991]
Alternative Column
The follow three columns have been confirmed toyield results for five test chemicals that are
essentially the same as the FUJIFILM Wako
column.
OSAKA SODA, CAPCELL CORE C18 column (2.7μm, 3.0 × 150 mm) [Cat. 51112]
ChromaNik, SunShell C18 (2.6 μm, 3.0 × 150 mm)[Catalog # CB6371]
Waters, CORTECS C18 Column (2.7 μm, 3.0 × 150mm) [Catalog # 186007373]
Agilent, Poroshell 120 EC-C18 (2.7 μm, 3.0 × 150mm) [Catalog # 693975-302]

Column Temperature

40ºC

Test chemical solution Temperature

25ºC
If the auto-sampler has a cooling function, test
chemical solutions can be kept more stable at 4ºC.

UV detector

Photodiode array detector ( for example, Shimadzu
SPD-M20A) or absorbance detector (281 nm)

Fluorescence detector

Fluorescence detector (for example, Shimadzu
RF-20A)

Injection Quantity

10-20 μl (The injection quantity varies according to
HPLC system. If peaks are too broad, the injection
quantity should be decreased.)

Run Time

20 minutes

21

Flow Conditions

NAC flow conditions
Time
0 min
9.5 min10 min13 min13.5 min20 minFlow
0.3 ml/min
0.3 ml/min
0.3 ml/min
0.3 ml/min
0.3 ml/min
End run

%A
70
45
0
0
70

%B
30
55
100
100
30

%A
80
55
10
10
80

%B
20
45
100
100
20

NAL flow conditions
Time
0 min
9.5 min10 min13 min13.5 min20 minFlow
0.3 ml/min
0.3 ml/min
0.3 ml/min
0.3 ml/min
0.3 ml/min
End runNote 1: The mixer quantity should be verified and adjusted in advance because the appropriate elution
pattern of NAC/NAL peak will not be shown if the mixer quantity for mixing each mobile phase is not
appropriate (For example, 0.5 ml mixing quantity is appropriate for Shimadzu prominence HPLC
system).
Note 2: The inner diameter of pipe and the length of pipe from column outlet to detector inlet must be lessthan 0.18 mm and less than 50 cm, respectively, because the peak of NAC/NAL might be broadened
depending on inner diameter and length of pipe
Note 3: If more than one wavelength is detected, also 291 nm besides 281 nm should be detected to checkout peak purities of NAC/NAL.
Graphical Display of Gradient for NAC/NAL Analysis
For NAL analysis

100
90
80
70
60
50
40
30
20
10
0

0.0

100
B.Conc [%]B.Conc [%]For NAC analysis

80
60
40
20
0

2.5

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

5.0 7.5 10.0 12.5 15.0 17.5 20.0

Time [min]Time [min]22Note: Visual inspection of test chemical solutions must be conducted prior to HPLC analysis. Generally,
precipitation is not a problem. However, if precipitation is observed, this should be noted in the datareporting template. Test chemical solutions may be centrifuged at low speed (100–400g) in vial to force
precipitate to the bottom of the well as a precaution, since large quantities of precipitate may clog the
HPLC tubing or columns. If more than 100 μl of supernatant can be recovered, in may be analyzed after
transfer to another plate. If precipitation removal is difficult to achieve by centrifugation or recovery of
the supernatant, filtration is available. In this case, it is necessary to confirm in advance that there is nochange in peak area of NAC and NAL before and after filtration using a reference control or standard
solution. Precipitate formation, removal, and transfer must be recorded and reported.
5-2.HPLC Sample Analysis Sequences
Each sample of HPLC analysis should be analyzed in number order below. Refer to the table showing
Examples of HPLC Sample Analysis Sequences for more practical sequences about HPLC analysis.
1. Start to analyze calibration standards and Reference Control A (N = 3).
2. The Co-elution Control does not need to be analyzed by turns if it is analyzed after analysis of standard
solution and Reference Control A.
3. Reference Control B should be analyzed three times (total six times) before and after the analysis ofsample, Reference Control C and Positive Control.
4. The Reference Control C, Positive Control and Test chemical solutions are analyzed. (After the first setof replicates of each sample is analyzed, the second set of replicates of each should be analyzed)
Example of HPLC Samples Analysis Sequences(A more specific analysis sequence can be found at the end of the SOP)

STD7
STD6
STD5
STD4
STD3
STD2
STD1

(Buffer solution for dilution)†Ref. 8-1. 1)

Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3

Ref. 8-1. 1)

Co-elution Control 1
Co-elution Control 2
Co-elution Control 3･･･
Co-elution Control n

Ref. 8-4.

23

Reference Control B, rep 1
Reference Control B, rep 2
Reference Control B, rep 3

Ref. 8-2.

Reference Control C, rep1§†
Positive Control, rep1
Test chemical solution 1, rep1
Test chemical solution 2, rep1
Test chemical solution 3, rep1･･･
Test chemical solution n, repn

First set of replicatesRef. 8-1. 2), 8-1. 3), 8-2.

Reference Control C, rep2§†
Positive Control, rep2
Test chemical solution 1, rep2
Test chemical solution 2, rep2
Test chemical solution 3, rep2･･･
Test chemical solution n, rep2

Second set of replicatesRef. 8-1. 2), 8-1. 3), 8-2.

Reference Control C, rep3§†
Positive Control, rep3
Test chemical solution 1, rep3
Test chemical solution 2, rep3
Test chemical solution 3, rep3･･･
Test chemical solution n, rep3

Third set of replicatesRef. 8-1. 2), 8-1. 3), 8-2.

Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6

Ref. 8-2.

†

Start to analyze calibration standard immediately after addition of Reaction Fixing Solution andpreparation of dilution series of standard solution.
§
Analyze three replicates for Reference Controls C. These results are used to calculate the NAC/NAL
depletion in each solvent and to verify that solvent used does not affect NAC/NAL depletion.

6. DATA ANALYSIS & CALCULATIONS
The concentration of NAC/NAL is calculated from peak area of absorbance at 281 nm and/or
fluorescence at Ex 284 nm/Em 333 nm for each test chemical solution based on the calibration curve
derived from standard solutions Std 1 to Std7. NAC/NAL percent depletion is calculated by dividing
NAC/NAL peak area of each test chemical solution by mean peak area of Reference Control C.

24

6-1. Calculation of Peak Area of NAC/NAL
Integrate the appropriate peaks and determine peak area for standards, test chemical solution andcontrols. The peak area of each integrated peak must be reported.
6-2. Calculation of Concentration of NAC/NAL
6-2-1. Generate a linear calibration curve based on the concentration of standards and the peak area.
Suitable calibration curves must have an r2>0.990.
6-2-2. Calibrate the mean NAC/NAL concentrations in Reference Controls A and C, SD and CV. The
each mean should be 3.2–4.4 μM. The NAC/NAL concentration of Reference Controls A and C
must be reported.
6-2-3. Calculate the mean NAC/NAL peak area, SD and CV for the Reference Controls C (N=3) for
each solvent used. The each mean concentration should be 3.2–4.4 μM. However, if 5%
DMSO/Acetonitrile is selected as a solvent for test chemical, the mean of concentration of NAC
should be 2.8–4.0 μM.
6-3. Calculation of Peak Area of NAC/NAL
6-3-1. Calculate the mean NAC/NAL peak area for the six Reference Controls B and the three Reference
Control C in acetonitrile, SD and CV. The CV must be less than 10%.
6-3-2. Calculate the mean NAC/NAL peak area and CV for the three Reference Controls C. The CV
must be less than 10%.
6-4. Calculation of Percent Depletion of NAC/NAL
6-4-1. For the Positive Control and for each test chemical, calculate the Percent NAC/NAL Depletion ineach replicate from the NAC/NAL peak area of the replicate injection and the mean NAC/NAL
area in the three relevant Reference Controls C (in the appropriate solvent), using the following
formula.
Percent NAC/NAL Depletion (% depletion) = [1- (NAC/NAL Peak Area in Replicate
Injection/mean NAC/NAL Peak Area in Reference Controls C)]×100
6-4-2. The mean Percent NAC/NAL Depletion (Average score) of the three replicate determinations, SD
and CV should also be calculated and reported. Report results to one decimal place.

7. DATA REPORTING (FOR NAC AND NAL)
System Suitability- NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em 333 nm for fluoresence ofStandard and Reference Control B and C replicate.
- The linear calibration curve should be graphically represented and the r2 reported.
- NAC/NAL concentration (μM) of Reference Control A replicate.
- Mean NAC/NAL concentration (μM) of Reference Controls C replicate, SD and CV.
Analysis Sequence25

Reference Controls:- NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em 333 nm for fluoresence ofReference Control B and C replicate.
- Mean NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em 333 nm for fluoresence ofthe nine Reference Controls B (N=6) and C (N=3) in acetonitrile, SD and CV (for stability ofReference Controls over analysis time).
- For each solvent used, the mean NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em
333 nm for fluoresence of the three appropriate Reference Controls C replicate (for calculation ofPercent NAC/NAL Depletion).
- For each solvent used in this assay, the mean NAC/NAL concentration (μM) of the appropriate
Reference Control C replicate, SD and CV.
Positive Control (Phenyl acetaldehyde)- NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em 333 nm for fluoresence of each
replicate.
- Percent NAC/NAL Depletion of each replicate- Mean NAC/NAL Depletion of the three replicates, SD and CV.
For Each Test Chemical:- Solvent chosen- Appearance of precipitate in the reaction mixture at the end of the incubation time.
It must be reported if precipitate was re-solubilized or centrifuged.
- NAC/NAL peak area at 281 nm for absorbance and/or Ex 284 nm/Em 333 nm for fluoresence of each
replicate (for systems equipped with a PDA detector the peak area at 291 nm should also be reported).
- Percent NAC/NAL Depletion of each replicate.
- Mean of Percent NAC/NAL Depletion of the three replicates, SD and CV.

8. ACCEPTANCE CRITERIA
8-1. Acceptance Criteria for Amino acid Derivative Reactivity Assay Run
All criteria must be met for the whole run to be considered valid. If three criteria are not met, the run
must be repeated for all test chemicals.
System Suitability:
Calibration Linearity r2 > 0.990
Mean NAC/NAL concentration of Reference Controls A = 3.2–4.4 μM
Positive Control:
The mean Percent NAC/NAL Depletion value of the three replicates for phenyl acetaldehyde or squaric
acid diethyl ester must fall within the range reported in the following table (Based on mean ±3SD frombackground data):
26

Percent NAC Depletion

Percent NAL Depletion

Positive control

Lower Bound

Upper Bound

Lower Bound

Upper Bound

Phenyl acetaldehyde

30

80

70

100

Squaric acid diethyl ester

30

80

70

100

Maximum Standard Derivatives for Positive Control replicate:Standard Deviation for Percent NAC Depletion must be < 10%Standard Deviation for Percent NAL Depletion must be < 10%
Stability of Reference Controls over analysis time:For each solvent used, the mean of the NAC/NAL concentrations of the three appropriate Reference
Controls C = 3.2–4.4 μM. However, if 5% DMSO/Acetonitrile is selected as a solvent for test chemical,
the mean of concentration of NAC should be 2.8–4.0 μM, as it is known that concentration of NAC
decreases because of oxidation of SH group by DMSO.
Reference Controls C in the analysis sequence:
CV of NAC/NAL peak areas for the nine Reference Controls B (N=6) and C (N=3) in acetonitrile mustbe < 10%. Moreover, CV of NAC/NAL peak areas for the three Reference Controls C (N=3) in eachsolvent must be < 10%.
8-2. Acceptance Criteria for Each Test Chemical
All criteria must be met for the run to be considered valid for a particular test chemical. If these criteria
are not met, the run must be repeated for the test chemical.
Maximum Standard Deviation of test chemical solution replicates:
Standard Deviation for Percent NAC Depletion must be < 10%Standard Deviation for Percent NAL Depletion must be < 10%
8-3. Data Acceptance for Amino Acid Derivative Reactivity Assay
The average score should be calculated from depletions of NAC/NAL, and the test chemicals should be
predicted to be either a Sensitizer or a Non-sensitizer according to following table.
NAC/NAL prediction model
Average score

JudgementLess than 4.9%

Non-sensitizer

4.9% or higher

Sensitizer

If an average score for NAC depletion in a test chemical falls within the borderline range described
below, additional testing should be performed to confirm the validity of the prediction. If the result of thesecond test is not concordant with the first test, a third test should be performed to determine a prediction
for the test chemical by majority of the three test results.
27

NAC and NAL prediction model: 3.0% ≤ average score ≤ 10.0%
NAC only prediction model: 4.0% ≤ NAC depletion ≤ 11.0%
8-4. Handling of Co-elution
If co-elution is observed in either the UV detector or fluorescence detector, the depletion measured by
the detector in which co-elution is not observed can be adopted. If co-elution is observed in both detectors
or if the fluorescence detector is not ready, handling of co-elution is described below.
8-4-1. Co-elution : Interference(1) Some test chemicals will co-elute with the NAC or NAL. In order to detect possible co-elution of
the test chemicals with NAC or NAL, the test chemicals included in the run must be injected alone(“Co-elution Controls”) at the beginning of the run sequence and their chromatograms comparedto the chromatograms of Reference Controls C in the appropriate solvent.
(2) If a chemical absorbs at 281 nm and/or fluoresces at 284 nm/333 nm (Ex/Em) and has a similarretention time as a NAC or NAL (overlap of valley-to-valley integration periods), then verify
whether or not the peak of test chemical is actually separated from the peak of NAC or NAL. If
the peak of test chemical is completely overlapped with the peak of NAC or NAL, and if theboundary of two peaks (valley between peaks) is located higher than baseline, co-elution of thetest chemical with that NAC or NAL should be reported. The “interfering” chemical peak shouldhave a peak area that is >10% of the mean NAC/NAL peak area in the appropriate Reference
Control. If co-elution occurs and proper integration and calculation of NAC/NAL depletion is notpossible, the data should be recorded as “interference” for NAC/NAL the chemical co-elutes with.
(3) Even if the test chemical does not co-elute with NAC or NAL, the Percent NAC/NAL Depletion
can appear to be < -10% if the concentration of Reference Control C is comparatively low.
Moreover, the Percent NAC/NAL Depletion can also appear to be < -10% due to inappropriate
handling of the measurement. In such cases, retesting of the test chemicals in question or otherappropriate measure should be taken.
8-4-2. Peak purity of NAC/NAL : Area ratio of 281/291 nm(1) When a Photodiode Array detector is used, co-elution of chemical and NAC/NAL may also beverified by looking at the UV spectrum at 291 nm in addition to 281 nm and calculating the area
ratio of 281/291. This value should be consistent over all test chemical solutions and standards fora distilled NAC/NAL peak and thus gives a measure of peak purity. For each test chemicalsolution, a ratio in the following range would give a good indication that co-elution has notoccurred. However, calculation of peak purity (area ratio of 281/291) might not always be possible,
particularly if the test chemical is highly reactive with the NAC/NAL leading to very small peaks.
90% < Mean Area ratio of Reference Control < 110%.
8-4-3. Co-elution : Depletion < -10%(1) If the Percent NAC/NAL Depletion is < -10%, it should be considered that this may be a situationof co-elution, inaccurate NAC/NAL addition to the reaction mixture or just baseline noise. If the28

NAC/NAL peak appears at the proper retention time and has the appropriate peak shape, the peakcan be integrated. In this case, there may just be baseline noise causing the NAC/NAL peak to bebigger or there may be some co-elution/overlap in retention time of the NAC/NAL and test
chemical.
(2) The calculated %-depletion should be reported as an estimate. In cases where a test chemical
co-elutes with NAL, the NAC only prediction model can be used. In cases where a test chemical
co-elutes with both NAC/NAL, the data should be reported as an inconclusive.
(3) In cases where the test chemical co-elutes with the NAC and the peak of NAC cannot be integrated,
the skin sensitization of test chemical cannot be predicted from the NAL depletion alone, and thedata should be reported as inconclusive.
8-4-4. Calculation of peak area for co-elution(1) If the peak of NAC/NAL and the peak of test chemical partially overlap, the peak area of
NAC/NAL should be integrated from valley of both peaks to baseline vertically.
(2) If the peak of NAC/NAL and the peak of test chemical completely overlap, the data should bereported as an Inconclusive, and the peak area should not be calculated.
8-4-5. Estimated depletion values
In some cases, a test chemical might co-elute with NAC and/or NAL though the test chemical reactwith NAC and /or NAL. If this is the case, co-elution will make the peak area of NAC/NAL appear to belarger than it really is, therefore the calculated percent depletion may be lower than the true value. Whenthe overlap in retention time between the test chemical and NAC/NAL is incomplete, percent depletioncan still be calculated with a notation of “co-elution – percent depletion estimates”. If the average scoreis below the criteria, the result should be reported as Inconclusive. However, unless NAC co-elutes withtest chemical, the NAC-only prediction model should be used.

Average score

No co-elution

Co-elution with NAC
alone or NAC and
NAL

Less than 4.9%

Non-sensitizer

Inconclusive

Apply NAC-only
prediction model

4.9% or higher

Sensitizer

Sensitizer

Apply NAC-only
prediction model

NAC only prediction model
NAC Depletion

Judgement

less than 5.6%

Non-sensitizer

5.6% or higher

Sensitizer

29

Co-elution with NAL
only

Example HPLC Analysis
There are 5 test chemicals. Chemical 1,2 and 3 are soluble in acetonitrile. Chemical 4 and 5 are soluble
in distilled water.
The following 96-well Microwell Plate should be set up:
Std 7 (Dilution buffer blank)
Std 6
Std5
Std5
Std4
Std 3
Std 2
Std1
Reference Control A, rep 1 (made with acetonitrile)
Reference Control A, rep 2 (made with acetonitrile)
Reference Control A, rep 3 (made with acetonitrile)
Co-elution Control for Chemical 1
Co-elution Control for Chemical 2
Co-elution Control for Chemical 3
Co-elution Control for Chemical 4
Co-elution Control for Chemical 5
Reference Control B, rep 1 (made with acetonitrile)
Reference Control B, rep 2 (made with acetonitrile)
Reference Control B, rep 3 (made with acetonitrile)
Reference Control C, rep 1 (made with acetonitrile)
Reference Control C, rep 1 (made with distilled water)
Phenyl acetaldehyde, rep 1
Chemical 1, rep 1
Chemical 2, rep 1
Chemical 3, rep 1
Chemical 4, rep 1
Chemical 5, rep 1
Reference Control C, rep 2 (made with acetonitrile)
Reference Control C, rep 2 (made with distilled water)
Phenyl acetaldehyde, rep 2
Chemical 1, rep 2
Chemical 2, rep 2
Chemical 3, rep 230

Chemical 4, rep 2
Chemical 5, rep 2
Reference Control C, rep 3 (made with acetonitrile)
Reference Control C, rep 3 (made with distilled water)
Phenyl acetaldehyde, rep 3
Chemical 1, rep 3
Chemical 2, rep 3
Chemical 3, rep 3
Chemical 4, rep 3
Chemical 5, rep 3
Reference Control B, rep 4 (made with acetonitrile)
Reference Control B, rep 5 (made with acetonitrile)
Reference Control B, rep 6 (made with acetonitrile)
Percent depletion for chemicals 1,2 and 3 is calculated based upon the mean NAC/NAL peak area of theReference Control C which are prepared with acetonitrile.
Percent depletion for chemicals 4 and 5 is calculated based upon the mean NAC/NAL peak area of theReference Controls C which are prepared with distilled water.

31