Waters LCMs Certified saMpLe ViaLs

Waters Corporation • 34 Maple StreetMilford, Massachusetts 01757-3696 USA508-478-2000 • Fax: 508-872-1990www.waters.com

[ White paper ]

We characterized the chemicals used in manufacturing by reversedphase

HPLC with UV detection, with the intention of developing a test to detect

residues or contaminants in the final product. The final test is run using

product packaged for a minimum of four days. The test detects residues

from manufacturing or contaminants released from the plastic wrap or

packaging. An active glass surface will pick up contamination from the

packaging very quickly. The test detects known processing residues at the

pp level.

Waters LCMS Certified Vials

In 2006, Waters® LCMS Certified Vials join our product line. To produce the

best product for LC/MS applications, we first surveyed vials from around

the world, including our current LC and GC certified vials. We then tested

all products, including components not currently in use, by the same

testing protocol.

Figure 1: Flow path of a typical sample through an entire analysis scheme

Background

The figure above shows a simple analysis scheme, beginning with some

form of sample preparation of the dissolved sample. The prepared sample

is then put into a vial and placed in the LC auto sampler. In HPLC the

sample is separated on the column and passes through an LC detector

for analysis. In LC/MS, after separation on column, detection and further

analysis are accomplished in the mass spectrometer. MS instruments

commonly used with LC are equipped with an atmospheric pressure

ionization interface [API]. Among various API interfaces currently

available;, electrospray ionization [ESI] is the most commonly used

technique.

2

Waters LCMS Certified Sample Vials

Waters Auto Sampler Vials

Waters is the leading manufacturer of analytical LC and LCMS instrumen-

tation and associated consumable products. Being in direct contact with

our customers, we understand the importance of auto sampler vials to the

performance of analytical instrumentation. There are many factors to

consider in selecting the proper vial such as:

• Needle design

• Auto sampler tray design

• Chemical compatibility

• Sample volatility

• Sample volume

• Chemical cleanliness

At Waters we take all of these factors into consideration in the design,

manufacture and delivery of our vials and accessories.

Why Certified Vials?

• Vials are manufactured by glass artisans and engineers who don’t

understand the requirements of auto samplers

• We understand the requirements for HPLC, specifically with respect to

auto samplers and detectors. As a leading producer of mass spectrom-

eters, we also understand the demanding requirements of MS.

You need to produce dependable results. Your time is too valuable to be

chasing sources of contamination or ion suppression. For these reasons, we

have introduced two lines of vials certified for their intended use.

LC/GC Certified Vials

Waters introduced LC/GC Certified Vials in 2004. In this certification pro-

gram, we covered dimensional specifications and chemical cleanliness.

We reviewed chemicals used in all phases of manufacturing to ensure

there were no residues or contaminants to interfere with your assays. The

chemicals used in manufacturing or packaging are:

• Surfactants used to prevent glass scratches and breakage

• Lubricants used on machinery

• Antistatic, antioxidants or mold release agents that may be presen in

plastic wraps or packaging

• Low MW silicone polymers from septa improperly cured

[ White paper ]

Separation A MS AnalysisSample Preparation

Extraction- Liquid-liquid- Solid phase- Pre-concentration- Filtration- Dissolution

Chromatography- Reversed phase- Normal phase- Ion exchange

Mass Spectrometry- Quadrupole- Time of Flight- Sectors (B & E)- FTICR

API Interface- ESI- APcI- APPI

Sample

Vials

Detection

Ultra-violetFluorescenceRefractive indexEvaporative light scattering

Result

3

As shown in Figure 2, the main function of the ESI source is to convert the

liquid effluent from the LC into a stream of charged droplets. Upon heating

and evaporation, the size of the droplet is reduced, and, consequently, the

density of charges at the droplet surface increases. The repulsion forces

between the charges increase until there is an explosion of the droplet.

This process repeats until analyte ions are freed from the droplet into the

gas phase. The ESI source is prone to phenomena called ‘ion suppression’

or ‘ion enhancement’ when the analytes of interest elute in the presence of

certain mobile phase additives or residual matrix components. Impurities

from any source may interfere with the ionization process. Impurities that

can be charged in MS affect the limit of quantitation [LOQ] or contribute to

a high relative standard deviation [RSD]. For more discussion on the topic,

please review cited papers.1,2 Vials are overlooked as a potential source

of problems. Yet they are an important link between the prepared sample

and the final analysis. If the vial is produced under poor quality control,

contamination from process [surfactants, lubricants] or human handling

[lotions, cigarette smoke] sources is possible.

Waters LC/MS Certification Process

We began by purchasing vials, caps and septa combinations from

around the globe and submitted all vials thru a screening protocol which

included:

• Soaking vials for 4 hours in different organic/water mobile phases

• Adding acid and base to some of the solvent combinations

• Analyzing the contaminants in these different solvents with ESI-MS

and recording the results. One hundred scans were combined for each

reference and sample.

The details of the experiment are included in a poster3 that can be

obtained by contacting Waters.

Results

In the following figures, we compare reference MS scans from the blank

wash solvents to those obtained from the same solvents soaking in

competitor’s standard vials and Waters LCMS Certified Vials. MS scans

from 250 to 1000 m/z for a competitor’s standard vial [top] and the

corresponding blank [bottom]. Note that the sensitivity of both scans is

identical. Clearly, this vial/cap/septum combination exhibits significant,

potentially interfering low mass contamination as well as clusters in the

high mass range. This is evidence of a range of various contaminants such

as oils, surfactants, and agents that bleed from the packaging material.

Such a vial is not suitable for LC/MS analysis3. This result is typical of the

vials we purchased from sources around the world. Though levels varied

widely, all the standard tested vials showed a significant contribution to

the MS background.

In Figure 4, a similar experiment is run using a Waters Certified LCMS

vial. Please note that the scales in Figure 3 and Figure 4 are identical.

If a vial is ‘clean’, i.e., not leaching any contaminants, its scan should be

similar to the reference. Clearly the vial analyzed in Figure 4 would be

considered clean and acceptable for use in LC/MS.

[ White paper ]

H+

Atmospheric Pressure Region

From LC[M+nH]n+

[M+nH]n+

To MassAnalyzer

MultiplyProtonated

Analyte

Droplet After Reductionin Size Due to Evaporation

“Coulombic Explosion”(Disintegration)

Vacuum

Ion Evaporation

ESI Needle 3-8 kV

N2

H+H+

H+

H+

H+H+

H+

H+H+H+H+

H+ H+

H+

H+H+H+

H+ H+

H+ H+H+

MM M

M

Figure 2. Electrospray Ionization Process

Scan ES+9.50e5

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

Scan ES+9.50e5

Competitor’s Vial

Reference

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z0

%

100

0

%

100

Figure 3: Determination of the MS background generated from a competitor’s vial

Bottom: MS background in the scan range from 250 to 1000 m/z. Top: MS data obtained on a competitive vial. One can clearly see the potential interference such a vial could cause in LC/MS analysis

This comparison shows that there are significant differences between

the cleanliness of vials from different vendors. Waters is working with

its suppliers to provide high-quality vials and septa with minimum

levels of contamination. These efforts are based on a research

program that identified the problem sources3. This in turn helps our

suppliers to eliminate potential for contamination in their manufactur-

ing processes.

Conclusion

In our survey of competitive products, we saw that the quality of vials,

when used for LC/MS applications ranged, from marginal to very poor

quality. Many of these vials are not suitable for LC/MS applications.

At Waters, our research into vials manufacturing and process controls

completed during the certification project for LC Certified Vials was

complimented by knowledge gained from our ongoing efforts in the

area of MS1.We have investigated signal suppression/enhancement

with various mobile phase modifiers and researched the best materials

of construction for sample prep devices with an eye toward extrac

ables and their impact on MS analysis2. Knowledge from the combined

experience in research, manufacturing and applications work has

been applied to the LCMS Certified Vials project. We have taken steps

to ensure proper selection of materials for LCMS Certified Vials and

the need to tightly controlled manufacturing processes and handling

procedures so that we can deliver a consistently clean vial for LC/MS

applications*. We will continue to provide the best products for your

demanding applications.

– Brian P. Murphy, Claude R. Mallet, Uwe D. Neue, Patrick D. McDonald

*All Waters LCMS Certified Vials are tested on a Waters® ZQ™ Mass

Spectrometer using MassLynx™ control. The vials come packaged with

a Certificate of Analysis showing the reference and vial scan for the

manufacturing lot.

Reference

1. C.R.Mallet, Z.Lu, J.R.Mazzeo, Rapid Commun. Mass Spectrom., 18: 49-58 [2004]

2. U.D. Neue and P.D. McDonald, Topics in Solid-Phase Extraction. Part 1. Ion Suppres-

sion in LC/MS Analysis: A Review. “Strategies for its elimination by well-designed,

multidimensional solid-phase extraction [SPE] protocols and methods for its quantita-

tive assessment,” Waters Whitepaper, 2005. Search for ‘720001273EN’ in Waters

Library on www.waters.com

3. C.R. Mallet, D. Diehl, J.R. Mazzeo, E.E. Chambers, “A Study of Contributions from

HPLC Vials to Ion Suppression/Enhancement in Electrospray Ionization,” Poster#

1270-20P, PittCon 2006, Orlando, 14 March 2006

Waters LCMS Certified Vial

Scan ES+9.50e5

Scan ES+9.50e5

Reference

250

0

0

%

%

100

100

300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

m/z

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Bottom: Blank

Top: Scan of the Waters LCMS Certified Vial

Figure 4: Experimental results typical of Waters LCMS Certified Vials

Waters and MassLynx, and ZQ are trademarks of Waters Corporation. All other trademarks are acknowledged. © 2008 Waters Corporation. All rights reserved.

September 2008 KK-PDF 720001517EN

Waters Corporation34 Maple StreetMilford, MA 01757 U.S.A.T: 1 508 478 2000F: 1 508 872 1990www.waters.com