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Article first published in Chromatography Today, May/ June 2018

Identifying Inherent Contamination in Deep Well Microplates /,,y Stephen Knight, Poroair Sciences Ltd, Wrexham, UK

Deep well micro plates are an important class of functional labware used for sample preparation, compound storage, mixing, transport and fraction

collection in many laboratories. They can also be used in many commercially-available autosamplers and come in a range of sizes and plate formats,

the most commonly used being 96-well and 384-well plates made from polypropylene.

In previous independently run evaluation

reports (1,2) in 2005 and again in 2013, it was

shown that commercially available deep well

microplates can harbour significant levels of

extractable compounds which come from

use of lower grade polypropylene polymer.

In this review additional findings by Weikert

et al. (3) will be considered. In this report

the researchers find significant levels

of contamination in samples of many

commercially available microplates.

The academic study by Howland performed

at Kent University broadened the scope of

previous work and tested not only "natural"

polypropylene microplates, but coloured

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and black plates and a much wider range

of manufacturer's products than before.

This study, gives data on a large range of

microplates from numerous manufacturers

based in Europe, USA and China. Mass

spectral data shows that persistent

contamination from a range of compounds

found in the raw polymer master batch

continues to be evident in many of the

microplates tested.

Background

The effect of extractables identified in this

report is complex and depends on the

exact application for which the plate is

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designed. However, it is clear that long chain

hydrocarbon contaminants will certainly

cause extraneous fluorescence signals in

any spectroscopic work and may even cause

false positives in large scale screens looking

at apoptosis or stasis in whole cells. In

forensics, these unwanted small molecules

can easily mask drugs of abuse or their

metabolites and cause ion suppression,

which leads to inaccurate quantification.

Scientists using deep well microplates must

ensure regular screening of the plates in

use if they are to avoid such unwanted

contamination. Testing can be carried out

in house, alternatively the use of certified

plates will ensure a certain quality standard.

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Figure 11: Plate M European 1ml round deep well ptate Figure 12: Plate Q USA 2ml square well deep well

MethodIn the 2005 study, samples of deep well

rnicroplate for testing were obtained

from all the major manufacturers. A new

unused plate was selected from each batch

and subjected to a stream of clean, dry

compressed air to remove any particulates

that may have accumulated. Testing

for polymer leachate and extractable

contamination was performed by incubating

overnight an appropriate volume of HPLC

grade methanol in three wells in each

sample plate. The methanol was spiked with

10 ug/ml of Caffeine as an internal standard.

The plates were sealed with a friction seal

and left to stand overnight.

After overnight incubation, 1 ul aliquots of

each well sample were subjected to analysis

on an Agilent GC-MS system using splitless

injection at 250°C.

Separation was performed on an Agilent

HP-5MS 0.25mm x 30m x 0.25um capillary

column using the following temperature

gradient.

Initial Temp 70°C Hold 2 min

Ramp 30C / min

Final Temp 310°C Hold 5 min

Detection was by positive ion EI-MS.

Integrated peaks were searched against a

NIST98 spectral library (scores over 95 being

significant hits in the database}. A blank was

also run using only caffeine-spiked methanol

held in silanised glass overnight which had

not contact with any polymer.

In order to simplify the full data set here,

results from each of the three wells per plate

tested have been combined and averaged.

ResultsPlates showing no measurable

contamination are shown in green. Some

minor contamination, defined as only one

contaminant and not necessarily in all

three wells tested, are in yellow. The worst

performing plates, showing considerable

contamination are shown in red. With

Description

Porvair 2rnl square well plate

Porvair 1 ml round well plate

Porvair 1 ml round well black plate

Porvair 1ml round well red plate

Porvair 1ml round well blue

European 1 .6ml low profile storage plate

USA 10ml storage plate

USA 2ml deep well plate

USA 1ml round well plate

USA 48 well deep well plate

China sourced 2ml deep well plate

European Microcentrifuge tubes, black

European 1ml round deep well plate

USA 2ml square well deep well plate

European 2ml square well, round bottomed plate

I.D.

D

E

F

G

H

A

B

J

N

C

I

L

M

Q

S

Results

Extractives Free

Extractibles Free

Extractibies Free

Extractibles Free

ExtractibSes Free

Minor Contaminant

Minor Contaminant

Minor Contaminant

Minor Contaminant

Significant Contaminant

Significant Contaminant

Significant Contaminant

Significant Contaminant

Significant Contaminant

Significant Contaminant

Plates/Tubes used in this study were from different sources of manufacturers in the USA and Europe.

s

Article first published in Chromatography Today, May/June 2018

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conducted by the SiO2

team used a more

aggressive extraction

method involving a

72 hour exposure to

acetonitrile and this

produced, albeit at lower

levels, more identifiable

contaminants from some

of the polypropylene

microplates under test.

These included anti-static

agents and antioxidants,

including the anti-

oxidant Tris (2,4-di-tert­

butylphenyl) phosphite,

slip agents which included

glycerol palmitate

and glycerol stearate

together with the polymer

nucleating agent bis­

(3,4-dimethylbenzylidene

sorbitol diacetal).

Figure 13: Plate S European 2ml square well, round bottomed plate

the caffeine standard giving a relative

abundance of 115000, any potential

contaminant used to group a plate into

green, yellow or red categories must show

an abundance greater than 10000. In the

worst case plates, contaminants exhibited

relative abundance in excess of 155000 and

in one case, off the scale of the integrator.

Contaminant Identification

The extracted contaminants were compared

against an NIST database to identify the

chemicals involved. These chemicals were

shown to be in a group which acts as plastics

enhancers and mould flow agents to assist

ease of injection moulding.

Other chemicals were apparent in some of

the products at lower levels and for the sake

of comparison to the original study were not

identified in full.

It is likely the above chemicals are

incorporated in the polymer mix at an early

stage to enhance ease of moulding and

fast cycle time of the mould to produce

the relevant products. The extra work

Contaminants

Eucrylamide

Dodecanoic Acid

Hexadecanoic Acid, methyl ester

Octadecanoic Acid, methyl ester

9-Octadecanoic Acid, oleic acid

Conclusion

The results show that, despite the ready

availability of high quality medical-grade

polypropylene for deep well microplate

production, more than half of the

tested plates exhibited some form of

contamination; typically, due to extractable

compounds leaching out of the plastic.

In both previous independent lab testing

studies, all Porvair Sciences deep-well plates

were shown to be contamination-free.

In the 2013 study, a significant selection of

deep well plates from different suppliers

were found to be contaminated with

extractables. This leads to the conclusion

that a low grade polypropylene was used

in the microplate production. Such low­

grade polypropylene often contains flow

modifier additives or "mould release

agents" which have been used to ease

the manufacturing process and help free

the moulded microplate from the mould

respectively. The use of methanol in this

study is significant. As a polar solvent it is

very good at dissolving small molecules

and has been shown to extract most

Commercial Use

Anti-Caking agent, lubricant

Mould release agent

Softner, accelerator activator

Plastics lubricant

Plastics lubricant, release agent

non-polymer bound compounds from

polypropylene microplates. It is also a very

suitable solvent for GC-MS. However, if an

even more powerful solvent, such as DMSO,

were used for this work, it is likely that even

more extractables would be seen.

Implications

With the continuing use of deep well plates

for sample introduction in LC/MS analysis

in pharma drug discovery, pesticide and

biocide development and detection, ADME/

Toxicology, forensics & narcotics testing and

in nee-natal errors of metabolism studies

where small molecule detection at low levels

is prevalent, inherent contamination from

the labware could pose a significant risk of

false positives or assay data corruption in

large-scale "hit-to-lead" screens.

Recommendations

It is recommended that deep well

microplates used for long-term storage

of samples in organic solvents are

regularly assessed to check for extractable

contamination. Consequently, it is important

that this phenomenon is understood before

stored compounds or samples are further

analysed. Efforts should be made to obtain

certificates of purity from the manufacturer

during method validation and where

this is not possible; extractables testing

should be carried out to prevent the risk

of contamination of valuable compounds

and samples. In the case of Porvair

Sciences plates, all the polypropylene

used is rigorously tested for extractables

and leachates before entering our clean­

room mould production. Care is taken

to use polyethylene bags which are also

extractable free and certificates of analysis

can be provided for each batch of product.

In addition the Class 10000 clean room

production environment ensures that the

plates are also DNA/RNA, DNA/RNAase and

endotoxin free at the point of use.

References

1. Extractables Testing in Deep Well Plates",

Ireland, Castleman et al, Porvair Sciences, 2005

2. Controlling inherent contamination in

deep well microplates", Knight & Castleman,

Porvair Sciences 2013

3. Plasma-treated Microplates with

Enhanced Protein Recoveries and Minimized

Extractables Weikart, Klibanov, Breeland,

Taha, Maurer and Martin, SLAS Technology,

2017, Vol 22(1)98-105

Extractable-free deep well plates from Porvair Sciences

96-well deep square44mm height

The family of 96 square well plates

was designed so that it would make the

interchange of plates simpler in

automated systems. All three plates have the same

geometry and the only variable is the well depth.

Deep well round 1 ml 42mm height

The plates are made in virgin polypropylene

to minimise extractables. Working volume is

1ml per well and total volume is 1.1ml.

Coloured plates aid identification when retrieving from storage.

Black plates are suitable for storing light sensitive compounds.

96-well deep square

96-well deep round 'common wall'45mm height

These revolutionary plates are made in

virgin polypropylene to minimise

extractables. Maximum volume is 2.075ml

per well and working volume is a useful 1.85ml, more than

any other comparable '2ml Round Well' plate in this class. The

'common wall' design allows the highest possible volume to

be used whilst maintaining an overall height of just 45mm,

complete with ANSI/SLAS standard base and footprint. Extra

working volume is thus assured in a convenient round bottom,

round well format.

384-well squarePorvair Sciences has a selection of

384-well polypropylene plates to

suit most applications. All of them

are made from pre-tested

polypropylene in Class 100000 clean rooms.

Description Well volume Sterile Use cap mat Quantity/case Cat. no.

Polypropylene, pyramid bottom 2.0ml 219004 50 219009

Polypropylene, pyramid bottom, sterile, inner bag of 5 2.0ml ✓ 219019 50 219027

Polypropylene, pyramid bottom, toughened for genomics 2.2ml 219004 50 219030

Polypropylene, pyramid bottom, toughened for genomics 2.2ml ✓ 219004 50 219031

Polypropylene, pyramid bottom 1.0ml 219004 50 219008

Polypropylene, pyramid bottom, sterile, inner bag of 5 1.0ml ✓ 219019 50 219026

Polypropylene, pyramid bottom 350µ1 219004 50 219006

Polypropylene, pyramid bottom, sterile, inner bag of 5 350µ1 ✓ 219019 50 219025

96-well deep round 'common wall'

Description Pk/Qty Part No.

96 deep well, 2ml/well Polypropylene round well rimless DNNRNAase free, inner bag of 5

96 deep well, 2ml/well Sterile Polypropylene round well rimless DNNRNAase free, inner bag of 5

Polypropylene storage plates round

50

50

219020

219021

Rim and bottom well shape Colour Well volume Sterile Use cap mat Quantity/case Cat. no.

Raised- round

Raised- round

No rim- round

Raised- round

Storage plates 384-well

Natural

Natural

Natural

Black

1ml

1ml

1ml

1ml

✓

219036

219042

219036

219036

50

50

50

50

219002

219012

219037

219412

Well shape, top & bottom Working well vol Sterile Qty/case Cat. no.

Square-round

Square-V

Square-V

58µ1

300µ1

300µ1 ✓

60

48

48

224001

219040

219041