6.3 fr ww asb 14-02 - sawis library · 2017. 5. 18. · buica_stander 3 this document is...

This document is confidential and any unauthorised disclosure is prohibited Version 2015

Industry allocated project number

PHI allocated project number

SATI

CFPA

SAAPPA/SASPA

DFTS

Winetech

[email protected] [email protected] [email protected] [email protected] [email protected]

Tel: 021 863-0366 Tel: 021 872-1501 Tel: 021 882-8470 Tel: 021 870 2900 Tel: 021 276 0499 X

___________________________________________________________________

FINAL REPORT

2016

1. PROGRAMME AND PROJECT LEADER INFORMATION Research

Organisation Programme leader

Research Team Manager

Project leader

Title, initials, surname Dr. A. Buica Dr. A. Buica Dr M Stander Present position Researcher

(Oenology) Researcher (Oenology)

Staff Scientist:CAF

Address DVO, Stellenbosch University

DVO, Stellenbosch University

Room 255

Tel. / Cell no. 021 808 9201 021 808 9201 021 808 5825 Fax 021 808 4781 021 808 4781 021 808 5825 E-mail [email protected] [email protected] [email protected] Co-worker Student

Title, initials, surname Present position Address Tel. / Cell no. Fax E-mail

2. PROJECT INFORMATION Research Organisation Project number

WW ASB 14-02

Project title Wine Quality Control: Screening of regulated substances

Short title

Fruit kind(s) grape Start date (mm/yyyy) 01/2014 End date (mm/yyyy) 12/2015

Key words Quality control; legal limits; screening

Approved by Research Organisation Programme leader (tick box) 3. EXECUTIVE SUMMARY

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This must report on the ENTIRE project. Address the objectives and milestones of the project as well as the impact of the study on the industry.

• The use of highly performant technologies and state-of-the-art equipment can help the

wine producers to obtain the certificates of analysis required in order to indicate

compliance for export.

• Combining existent methods will reduce the cost of these analyses while taking care not

to lose performance of the measurements. New methods, aimed at other high risk

compounds that will most probably be included soon in the list of regulated substances,

is also important to avoid delays associated with the development and implementation

and will save the producers the costs related to the disruption in exports

• Methods for the determination of preservatives (natamycin and sorbic acid), surfactants

(benzalkonium chlorides) and biogenic amines were developed and will be validated and

implemented with a focus on the biogenic amines

4. PROBLEM IDENTIFICATION AND OBJECTIVES State the problem being addressed and the ultimate aim of the project. There has been growing pressure on the SA wine industry to prove the quality of its products to

its overseas clients. Certificates of analysis for certain unwanted substances in wine (such as

natamycin, ochratoxin A and sorbic acid) are required by many producers in order to show

compliance with the exporting regulations. The costs of these analyses will grow as more

compounds are added to the list, which place a financial burden on the industry. It is important

to act in a pro-active way to develop sensitive, efficient and at the same time cost effective

methods to deal with this problem.

Currently most regulated banned substances in wine including natamycin, ochratoxin A and

sorbic acid have to be analysed separately and therefore at a higher cost than if the compounds

are analysed together.

The project will also investigate other high risk compounds sometimes present in wines like

phthalates, other mycotoxins like fumonisin, colourants, with a focus on biogenic amines such

as histamine, tryptamine and tyramine. It is possible that some of these compounds will be

included on the list of regulated substances in the near future. At that stage, being ready to

screen wine for these substances will avoid the delays associated with the development and

implementation of new methods and will save the producers the costs related to the disruption

in exports.

Liquid-chromatography mass spectrometry (LC-MS) is the technique of choice for trace level

determinations of these regulated compounds. The LC-MS conditions in the current methods for

sorbic acid, ochratoxin and natamycin are similar. The most differences are related to the

sample preparation. New sample preparation methods have to be developed that will enable the

accurate determination of these compounds. Different LC-MS analysis conditions have to be

evaluated to compensate for the new sample preparation procedures.

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In this project we seek to:

a) Establish a robust and fast routine screening method for regulated substances

b) Establish robust and fast routine methods for high risk substances (biogenic amines,

phthalates, colourants)

The chemistry of different regulated compounds vary to a large extend. The biggest risk we

predicted was that more methods are needed to test for all of them which will increase the cost

of the project and the time to finalise it. This risk is however minimal compared to the advantage

to the industry in analysis costs if even only two of the existing methods could be combined.

5. WORKPLAN (MATERIALS AND METHODS) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage conditions and examination stages and parameters. Add additional rows if required.

1. Method development for the sample preparation for target compounds (biogenic amines)

2. Method optimization for the LC-MS conditions for the analysis of the target compounds

in terms of speed of analysis and robustness

3. Method validation for both sample preparation and instrumental analysis to prove their

selectivity, accuracy and robustness

4. Method development and optimization for other target analytes (sorbic acid, ochratoxin,

natamycin, etc.)

5. Implementation: Screening of samples (young wines and from previous vintages)

6. RESULTS AND DISCUSSION

The development of sample preparation procedures and instrumental method(s) – various methods

have been developed or adapted to include a larger number of compounds of interest.

Natamycin and sorbic acid are now included in one single LC-MS/MS analysis instead of two. The limits

of quantitation and detection for both compounds are still within the levels required for wine

certification (the determination did not lose sensitivity by combining the two methods). This method is

already implemented at CAF.

The sample prep consists of a dilution of 1/1 of the sample with methanol and solid phase extraction on

amino cartridge. The range for detection is for natamycin less than 30 ppb and for sorbic acid 0.004 – 4

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ppm. For the analysis of sorbic and benzoic acid at higher concentrations in juices and mixer are done by

LC-HRMS.

In all these cases, the advantage is the simple sample prep, wide range of concentrations detected, and

short analysis time. What is still left as a challenge that could not be overcome until now, Ochratoxin

retained on the SPE cartridge during the sample prep step, so this compound cannot be yet analysed

with the rest in one single run.

Benzalkonium chlorides :12 and :14 are cationic surfactant agents from the quaternary ammonium

group. They can be determined in a single run by LC-HRMS in less than 12 min, in the range from 40 ppb

to 40 ppm, with the possibility of lowering the limit of detection if necessary. This method is also already

implemented at CAF.

Phthalates are a group if chemicals that are used as plasticizers (substances added to plastics to

increase their flexibility, transparency, durability, and longevity). Because phthalates are not chemically

bound to the plastics they're added to, they're continuously released into the air or food or liquid. They

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are considered dangerous for health as they act as endocrine disruptors (affect the normal behaviour of

hormones) and are associated with an increased risk of certain cancers. The table below presents some

figures of merit for the phthalate determination by GC-MS/MS (the method implemented at CAF) and

the legal limits for some of the components of the group.

As you can see, the legal limits are very high compared to the performance of the method. It should also

be noted that EC Regulation no. 10/2011 also includes a positive list, i.e. one of all authorised molecules

in materials that come into contact with foodstuffs. Those molecules that are not cited are prohibited,

and thus are not allowed.

Phthalates can be determined by LC-MS/MS, using as sample prep direct injection or dilution for high

ethanol containing samples. The challenge is related to issues with high background noise which can

lead to difficulties in quantifying low levels of compound. Also, as you can see from the chromatograms

below, DIDP and DINP (top two signals) have isomers, peak shape is not good, and this can lead to lack

of accuracy for the quantitation of these compounds.

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The alternative is to measure phthalates by GC-MS/MS. The sample preparation is relatively

strightforward, extraction with hexane. As you can see from the chromatogram below, the peak shape is

excellent (compounds are separated and peaks are very narrow). From the full MS scan more, the signal

for quantification of individual compounds can be extracted, as exemplified in the second

chromatogram (extracted ion 149 for DEPH at retention time 21.06 for example). Extracted spectra are

used for compound identity verification, in addition to the retention time. In the example given here, for

DEPH spectral confirmation with the two transitions that will be used in MS/MS mode: 279.17 → 167.05

and 149.03.

Buica_Stander 7


Below can be seen a snapshot of the GC-MS/MS software with the calibration table for DEPH (2.5-200

ppb), R2 of 0.9959. The list of all the compounds that can be analysed is on the right side.

This project was in fact initiated when we discussed the need for a method to analyse biogenic amines

of importance (mainly histamine, tyramine and tryptamine, but others are included in the method). This

offered us not only the challenge to develop a method in which these compounds can be determined in

less than 15 min, but also the opportunity to test some columns produced with new stationary phase

technology. Since biogenic amines are polar compounds, their separation can be achieved taking

advantage of the differences in the polarities of the different compounds. Regular columns, such as C18,

do not behave well under the conditions necessary for the separation of more polar compounds and

that behaviour reflects in bad peak shape (leading to difficulties in accurate measurement) and

sometimes even in the physical degradation of the stationary phase of the column.

For this particular application, we have tested a number of columns suitable for the separation of polar

compounds, with various rated of success. The first column tested was a BEH Amide column from

Waters. The structure of the stationary phase is described as “ethylene-bridged hybrid”. As you can see

from the figure below, peak shape was bad and noise very high for some of the compounds.

Buica_Stander 8


The other two columns evaluated during the method optimization were also from Waters, cHILIC (with a

phosphorylcholine stationary phase) and pHILIC (with a sulfobetaine stationary phase). One of the main

differences between these two types of stationary phases is the positioning of the charges in rapport

with the support of the stationary phase. This, in turn, affects how a polar compound (charged or not)

interacts with the stationary phase and the steric hindrance (how easily the analyte and the separation

active site fit). As you can see from the figure below, the peak shape is better (more symmetrical and

peaks are narrower in most cases) for the cHILIC stationary phase. Also the noise is lower for cHILIC. For

these reasons, cHILIC column was chosen for the next method step, namely validation.

The figures of merit for the validation are presented in the table below. Initially the sample prep

consisted of only filtering the sample and injecting directly. When investigating the matrix effect, we

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noticed that the response factors (as seen in the slopes of the calibration curves) were heavily

influenced by the matrix. This also reflected in the recovery of the compounds of interest in the red and

white wines, which were at unacceptably low levels especially for red wine.

In the end it was decided that, since the cause of the low response factor for red wine to a large extent

and for white wine, too, was the noise generated by the high concentration of other matrix

components, the best way to improve recovery was to dilute the samples. This did not affect the LOD

and LOQ, but it improved the recovery greatly, as seen in the table above.

For the final method, the sample prep consists of a 1/5 dilution of the sample with 50/50

acetonitrile/water containing 1.5% HCl, since the acid helps stabilize Histamine. As mobile phases,

Solvent A consists of 50 mM ammonium formate and Solvent B of acetonitrile:methanol:isopropanol

(49:49:2). The gradient starts with 50% B and is linear to 3% B using a flow rate of 0.3 mL/min.

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To test the method with real samples, we analysed a number of wines that were available at the

moment in the lab to see if there are any further issues. There weren’t any other problems and the

results are presented in the table below. The results are in mg/L.

A PCA representation of the data shows that, for the sample set analysed, there is no real differentiation

(grouping) between the biogenic amine composition of red and white wines. We will not hypothesize

further on this, since other (discriminant) tests can possibly indicate otherwise but we did not apply

them to this sample set.

Two peer reviewed publication as well as validated methods to be used by testing laboratories.

The commercial value in the project lies in the cost saving to industry in wine analysis. Conference participation(s) in the form of proceedings, posters and/or oral communications.

Buica_Stander 11


7. COMPLETE THE FOLLOWING TABLE

Milestone Target Date

Extension

Date Date

completed Achievement

1. Literature survey

April 2014 n/a April 2014 done

2. Consultation with industry and Dept of Agriculture

April 2014 n/a n/a The list proposed was not amended

3. Order chemicals October 2014 n/a done

4. Development sample preparation procedures and instrumental method(s)

October 2014 n/a April 2015 Done

5. Validate method(s)

October 2014 n/a April 2015 done

6. Determine which wines to be tested Source wines Test wine

November 2014-July 2015

n/a November 2014-July 2015

There were no special requests for the samples to be analysed

7. Journal publication(s) – final milestone

December 2015

December 2016

n/a In progress

8. CONCLUSIONS Even though not all the methods had to be developed from scratch (some were already available in the CAF portfolio), this project demonstrated that even methods that are already implemented in routine can be modified in such a way that, sometimes with minimal effort, higher throughput or lower costs per analysis can be achieved. In the case of biogenic amines, the method had to be developed fully, but that gave us the opportunity to test new column technologies that will be without a doubt useful in the near future for new applications. 9. ACCUMULATED OUTPUTS a) TECHNOLOGY DEVELOPED, PRODUCTS AND PATENTS The new, improved, or combined methods are already implemented at CAF for the use of the industry.

b) SUGGESTIONS FOR TECHNOLOGY TRANSFER At least one popular article should be published from this work, highlighting the novelty and the advantages of the methods.

c) HUMAN RESOURCES DEVELOPMENT/TRAINING

Buica_Stander 12


Student Name and Surname

Student Nationality Degree (e.g. MSc Agric, MComm)

Level of studies in

final year of project

Graduation date

Total cost to industry

throughout the project

Honours students

Masters Students

PhD students

Postdocs

Support Personnel

N.F. Hiten SA 0

M. Taylor 0

L Mokwena SA 0

d) PUBLICATIONS (POPULAR, PRESS RELEASES, SEMI-SCIENTIFIC, SCIENTIFIC) Buica A, Stander M A robust LC-MS/MS method for the quantification of biogenic amines in

grape-related matrices (in preparation) e) PRESENTATIONS/PAPERS DELIVERED Buica A, Stander M (2015) Wine quality control: screening for high risk compounds poster

presented at the 9th Symposium In Vino Analytica Scientia, Mezzocorona, Italy 2015 10. BUDGET

a) TOTAL COST SUMMARY OF THE PROJECT

YEAR

CFPA DFTS Deciduous SATI Winetech THRIP OTHER TOTAL

2014

135 000 40 000 175 000

2015

135 000 40 000 175 000

b) FINAL BUDGET/FINANCIALS OF PROJECT

Project duration Proposed

budget

Actual cost

incurred Variance Notes

TOTAL INCOME

Industry Funding 270 000

PHI Funding

Buica_Stander 13



budget

Actual cost


Other Funding 90 000 The project was submitted to

THRIP as part of a bigger

group of projects and as

such it is difficult to estimate

how much of the funding was

for this particular project, the

ratio was 1/3

TOTAL EXPENDITURE

Running Expenses

General operating costs

(printing, communication, etc.)

5 000 5 000 0 Incl. Printing and shipping of

poster to IVAS, Italy

Local Travel 20 000 10 000 +10 000 No participation to local

conferences in 2015

Publication costs 8 000 8 000 0 The article hasn’t been

published yet, the amount is

based on an approx. 500

Euro publication fee

Lab Analysis 80 000 96 800 -16 800 Includes CAF lab fees and

operator time, SU admin

fees

Lab Consumables 175 000 175 000 0 Vials, caps, columns for the

new applications, solvents,

SPE cartridges for sample

prep method development,

etc.

Other 35 000 35 000 0 Standards

Running expenses SUB-

TOTAL

323 000 329 800 -6 800

HR Administration and Project

Management

HR Technical

HR Research

Student Bursaries

HR SUB-TOTAL

OTHER EXPENSES

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budget

Actual cost


20 000 25 000 -5 000 Administration costs SU –

transfers between DVO and

CAF

SURPLUS / DEFICIT 343 000 354 000 +6 000 surplus

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