WHY ARE LC VIALS SHOWING GHOST PEAKS WITH THE NEW GENERATION OF MASS SPECTROMETERS? Claude R. Mallet1, Brian P. Murphy2

Workflow Integration Group1

Chemistry Business Unit2

CHAPTER 2 LEACHABLES FROM SILICON SEPTUM: CHROMATOGRAPHY

2

INT RODUCT ION

In this second installation of leachables experiment, we introduce

the Open Architecture UPLC System with 2D-LC Technology1,2

and at-column dilution. This technique demonstrates low-cost

enrichment of sample, followed by chromatography, allowing the

user to screen a large number of samples in a short time frame.

The example is vials and septa. Packaging material leaching into

contents is a common concern in many industries; for example,

pharmaceuticals and food, to name two. This technique and

instrumentation allows a user to quickly screen materials to select

appropriate packaging for the end application.

EX PERIMENTAL

For the enrichment analysis, the Open Architecture UPLC System

with 2D-LC Technology was upgraded with the at-column dilution

option1. The chemistries used for D1 and D1 were the Oasis HBL

20 µm (2.1 x 30 mm) and the BEH C18 1.7 µm (2.1 x 50 mm)

columns, respectively. The loading conditions used for at-column

were set at 5% dilution (loader pump at 0.2 mL/min & dilutor

pump at 4 mL/min). The injection volume was set at 500 µL for a 4

min loading time.The trapped analytes were back flush eluted with

a 0.5 mL/min gradient. The elution started at 5% to 95% organic

for 5 minutes with 0.5 % formic acid. Three organic modifiers

were used for the chromatography (methanol, acetonitrile, and

acetone). The mass spectrometer was set under scan mode (100

to 1000 amu) with positive electrospray (ESI). Each of the 2-mL

silicone cap extracts (water, methanol, acetonitrile, and acetone)

were subjected to all three chromatography conditions. The 2-mL

vial leachable experiments were conducted with the same protocol

with one exception. The vials were covered with an aluminum foil

to remove the potential contribution of the septum cap.

RESULT S

Given the infusion results presented in Chapter 1, the next step

was to evaluate the chromatography behavior. Using the Open

Architecture UPLC System with 2D-LC Technology upgraded with

at-column dilution, the results for the polyethylene cap, low bleed

silicone, and a prototype silicone (acetone extract) are shown in

Figure 1a, 1b, 1c, 1d, 1e, and 1f, respectively. In each figure, a

total ion chromatogram (TIC), with its corresponding combined

spectrum, showcases the chromatography profile for the MeOH,

ACN, and Acetone gradient. From each spectrum, an extracted

chromatogram was produced for ion with the highest intensity.

Also, a second extracted chromatogram was generated for a

common ion, in this case the 609.8 from the silicone distribution.

MS2 ES+

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 TIC2.01e10

MS2 ES+ TIC

2.01e10

MS2 ES+ TIC

2.01e10 LC MeOH

LC ACN

LC Acetone

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100 MS2 ES+

1.63e7 370.86 148.84

222.97 354.86 301.03

296.88

428.85

502.77

723.56 517.48 576.76

650.84 780.84 796.68 888.77

MS2 ES+ 1.07e7 131.49

494.53 453.49 148.92

370.86

182.91 282.16 184.83 354.94 444.85 708.12 664.36

535.64 606.36 722.44

775.80 849.89

MS2 ES+ 6.94e7 137.96

196.10 778.12 609.88 370.86 282.08 428.85 502.77 683.96 831.81

Figure 1a. TIC and combined spectrum for methanol vial extracts.

3

From the TIC and the extracted chromatograms, it becomes

apparent that after the entity’s molecular weight is identified,

understanding the chromatography behavior is also key for

effective identification of potential ion suppression zone

during elution. With the polyethylene cap, the 370.8 ion shows

an intense signal with an acetone gradient when compared

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ TIC

3.16e10

MS2 ES+ TIC

3.16e10

MS2 ES+ TIC

3.16e10

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100 MS2 ES+

6.58e7 137.96

338.63 196.10 321.19

274.96 370.86 609.96 428.85 502.77 778.20 683.88 831.81

MS2 ES+ 1.48e7 338.15

131.49

148.92 321.19

182.83 313.11

494.61 339.03 453.57

444.93

609.88 535.72

536.76 610.76 611.96 685.08

693.40 775.96 803.96

MS2 ES+ 4.16e7 338.15

148.92 300.95 222.97

162.83

341.11

428.77 444.85

502.77 518.84 675.24 831.73 779.00 889.65

LC MeOH

LC ACN

LC Acetone

to acetonitrile and methanol. The corresponding extracted

chromatogram at 370.8 shows a minor signal with the acetone

gradient. However, the 370.8 shows a well resolved distribution

with the acetonitrile gradient suggesting that the 370.8 has a

high solubility affinity with acetonitrile. The retention time at

the end of the gradient suggests a non-polar nature, since the

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ 370.8

1.02e9

MS2 ES+ 370.8

1.02e9

MS2 ES+ 370.8

1.02e9

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ 609.8

1.55e9

MS2 ES+ 609.8

1.55e9

MS2 ES+ 609.8

1.55e9

Figure 1c. TIC and combined spectrum for acetonitrile vial extracts.

Figure 1b. Extracted mass chromatograms for methanol vial extracts

4

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ TIC

8.10e10

MS2 ES+ TIC

8.10e10

MS2 ES+ TIC

8.10e10

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100

m/z 100 200 300 400 500 600 700 800 900

%

0

100 MS2 ES+

6.40e7 137.96

370.86 354.94

338.95 223.05

296.96 428.85

502.85 609.80 504.69

683.80 650.76

759.72 777.96 849.89

MS2 ES+ 3.24e7 683.80

609.88 535.88 354.94

321.91 222.97

131.33 190.02

502.93 428.77

429.81

537.80

538.68

610.76

612.68

684.76 685.72

757.88

687.72 760.76 761.72

785.88 849.89

MS2 ES+ 6.71e7 428.77

354.86

236.89

221.05 296.96

429.57 683.80 430.77

502.85 504.69

576.76 614.68

685.72 688.68

689.64 831.81 706.60

757.80 836.77

LC MeOH

LC ACN

LC Acetone

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ 609.8

1.66e9

MS2 ES+ 609.8

1.66e9

MS2 ES+ 609.8

1.66e9

Time 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

%

0

100 MS2 ES+ 338.6

1.61e9

MS2 ES+ 338.6

1.61e9

MS2 ES+ 338.6

1.61e9

Figure 1e. TIC and combined spectrum for acetone vial extracts.

Figure 1d. Extracted mass chromatograms for acetonitrile vial extracts.

5

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separation was performed on a silica-C18 sorbent. The 609.8

ion also shows a late-elution profile, while the high signal and

Gaussian distribution with the acetone gradient suggest the 609.8

ion has a stronger affinity for intermediate polarity solvent. The

low bleed silicone cap shows a similar distribution for the 609.8

when compared to the polyethylene cap. However, the 338.6 ion

shows a chromatography profile with a Gaussian peak shape by

using an intermediate polar solvent. As seen in Figure 1c, the

methanol gradient produced a distorted signal. However, with

acetonitrile, the 338.6 ion produced a sharp rise with a prominent

peak tailing, suggesting a better affinity with acetonitrile than

methanol. With respect to the prototype silicone cap (see Figure

1f), the chromatography profile for the 370.8 ion shows similarity

with the polyethylene cap. The intense signals in all three extracted

chromatograms clearly indicate a massive leaching effect with

significant matrix effect. The 609.8 ion shows similar result.

References.

1. Mallet, C., Multi-Dimension Chromatography Compendium: Trap and Elute vs. AT-column dilution, Waters Corporation, 2014. 720005339en.

2. Mallet, C. Why are LC Vials Showing Ghost Peaks with the New Generation of Mass Spectrometers? Chapter 1 – Leachables from Silicon Septum: Infusion Analysis. Waters Corporation, 2014. 720005335en

CONCLUSIONS

In this application, leachable experiments were conducted with

minimum manual labor. The Open Architecture UPLC System

with 2D-LC Technology with infusion and at-column dilution

configurations enabled 500:1 enrichment analysis by using

large-volume injection (aqueous and organic). These two

configurations eliminated the time-consuming evaporation-to-

dryness and reconstitution steps. Overall, the results indicated a

wide range of leachable entities in septum and vials; therefore,

trace level analysis (low ppt range) now has to deal with these

significant contributions.

Time 4.00 6.00 8.00 10.00 12.00

%

0

100 4.00 6.00 8.00 10.00 12.00

%

0

100 4.00 6.00 8.00 10.00 12.00

%

0

100 MS2 ES+

370.8 1.49e9

MS2 ES+ 370.8

1.41e9

MS2 ES+ 370.8

1.55e9

Time 4.00 6.00 8.00 10.00 12.00

%

0

100 4.00 6.00 8.00 10.00 12.00

%

0

100 4.00 6.00 8.00 10.00 12.00

%

0

100 MS2 ES+

609.8 1.74e9

MS2 ES+ 609.8

1.67e9

MS2 ES+ 609.8

1.72e9

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©2015 Waters Corporation. Produced in the U.S.A. April 2015 720005337EN AO-PDF

Figure 1f. Extracted mass chromatograms for acetone vial extracts.