Low-extractable Millex Samplicity® filters are ideal for preparing samples for sensitive HPLC detectors

The Samplicity® filtration system was developed to filter up to 8 samples directly into standard HPLC vials using vacuum-based filtration. The samples can then be used in downstream HPLC analysis. Given the high sensitivity of high performance liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) analyses, it is critical to ensure that the filtered sample is not contaminated with extractables.

This application note reports the results of tests for extractables from Millex Samplicity® LCR (0.45 µm hydrophilic PTFE) and LG (0.20 µm hydrophilic PTFE) filters using three commonly used HPLC detectors. The detectors used were an ultraviolet/visible (UV-Vis) spectrophotometer, a mass spectrometer and a fluorescence detector.

Materials and Methods

Samplicity® Filtration System. 0.45 µm and 0.2 µm Millex Samplicity® hydrophilic PTFE filters (Cat. Nos. SAMPLG001 and SAMPLCR01) were used with the Samplicity® filtration system (Cat. No. SAMPSYSGR (green) or SAMPSYSBL (blue)).

Extraction. Four or eight Millex Samplicity® filters were set on the Samplicity® filtration system and 1 mL of appropriate solvent (Milli-Q® water, acetonitrile (Cat. No. A955-4), methanol (Cat. No. A466-4), tetrahydrofuran (Cat. No. T425-1) or acetone (Cat. No. A949-4)) was added to the filter funnel. Sample was filtered and collected in standard (12 X 32 mm) HPLC vials. This extract was collected and labeled as the 1st mL extract. An additional 1 mL of solvent was then added to the same filters and extracts were collected in another set of clean standard HPLC vials. This extract was labeled as the 2nd mL extract. The extracts were analyzed by HPLC separation followed by the various detectors.

HPLC. Samples were injected (100 µL per injection) and resolved on a Waters µBondapak® C18 column, 3.9 mm x 300 mm, 10 µm, 125 Å. Mobile phase A was Milli-Q® water, and mobile phase B was acetonitrile. Flow rate was 2 mL/min. HPLC gradient profile is shown in Table 1.

Table 1.

HPLC gradient profile.

Time, min % A % B
0 100 0
10 0 100
13 0 100
16 100 0
20 100 0

Table 2.

Mass spectrometry detector settings

Q3 Scan 100-1500 m/z in 5 seconds
Source Turbo ion spray
Curtain gas 20
Ion spray voltage 5000
Temp OFF
Ion Source gas 1 20
Ion Source gas 2 0
Interface heater ON
De-clustering potential 20
Focusing potential 200
Entrance Potential 10.0

Figure 1.

HPLC-UV extractables pro-file for Millex Samplicity® 0.20 µm Hydrophilic PTFE filters (acetonitrile as extraction solvent). The various traces represent extracts from replicate filter units.

HPLC-UV extractables pro-file for Millex Samplicity® 0.20 µm Hydrophilic PTFE filters (acetonitrile as extraction solvent). The various traces represent extracts from replicate filter units.

Figure 2.

HPLC-UV extractables pro-file for Millex Samplicity® 0.45 µm Hydrophilic PTFE filters (acetonitrile as extraction solvent). The various traces represent extracts from replicate filter units.

HPLC-UV extractables pro-file for Millex Samplicity® 0.45 µm Hydrophilic PTFE filters (acetonitrile as extraction solvent). The various traces represent extracts from replicate filter units.

UV Detection. UV radiation-absorbing extractables were detected using a Waters 2489 UV Detector, Model # 87E, with three detection wavelengths: 214 nm, 254 nm and 280 nm.

Fluorescence Detection. Fluorescent extractables were detected using a Waters 2475 Fluorescence Detector, Model # 475. Excitation wavelength was 490 nm; emission wavelength was 525 nm.

MS Detection. Mass spectrometry of extracted samples was performed using a AB SCIEX API 2000™ Triple Quad MS/MS Turbo Spray source, using ESI + conditions. Detailed settings are listed in Table 2.

Results and Discussion

UV Extractables Analysis. The Samplicity® system is validated for sample preparation for HPLC. Therefore, Millex Samplicity® 0.45 µm and 0.20 µm PTFE filters are certified for HPLC cleanliness using standard HPLC-UV analysis. The HPLC certification ensures users that the filter will not introduce any extractable impurities into the sample. Representative LC-UV chromatograms show that no detectable extractables were leached from Millex  Samplicity® 0.45 µm and 0.20 µm PTFE filters using acetonitrile as an extraction solvent (Figures 1 and 2). Analysis of acetonitrile extracts using multiple replicate filter units yielded superimposable traces, showing that the results were highly reproducible.

The peak shown at ~ 11 min. is present in blank acetonItrile as well, and hence it is not considered an extractable peak originating from the Millex Samplicity® filters. Of the 3 wavelengths tested, typically the lowest wavelength (214 nm) shows the most absorbance. Similar UV extractables profiles were seen when using Milli-Q® water or methanol as extraction solvents.

LC-MS Extractables Analysis. LC-MS has not only become an extremely common analysis technique in pharmaceutical and biotechnology laboratories, but it is also being used in various other industries. Mass spectrometry offers highly sensitive and specific analysis of very low concentration analytes present in the sample. This means that any extractables that show up in the sample will affect the analysis and impact quantitation of analytes.

Determining MS extractables can be very challenging,   because the conditions under which extractables are  measured greatly affect the calculated level of extractables. In this study, we only used the ESI + ionization mode, and therefore may have missed some extractables which are only ionizable under ESI – / APCI ionization mode or are completely non-ionizable. We also compared the LC-MS extractables results with those of a commercially available MS-certified syringe filter (non-Millipore) that is reported to have very low levels of extractables.

With water and acetonitrile as extraction solvents, all the three filters showed very similar levels of low extractables; however, with methanol as extraction solvent, the LC-MS certified syringe filter showed a much higher level of extractables, which can affect the downstream LC-MS analysis. Table 3 shows the highest observed total ion current for a given solvent and filter type; values were representative of 2-4 replicate filter units tested. Figure 3 shows the average LC-MS methanol extractables profile for each of the filters tested as well as methanol blank.

HPLC-Fluorescence Detection. Next to UV detection, fluorescence detection is the second most frequently used detection method used in HPLC analysis. Fluorescence detectors are very selective and sensitive, enabling detection and quantitation of analytes at levels close to few ng/mL with very low background noise. What this means from a sample preparation perspective is that the filter used for preparing samples prior to HPLC analysis should introduce minimal extractable impurities as detected by a fluorescence detector. Similar to the challenges with LC-MS, fluorescence intensity varies based on the excitation and emission wavelengths. In this study, we decided to choose the excitation and emission wavelengths that correspond to fluorescence properties of fluorescein. The emission efficiency of this fluorochrome is very high, facilitating highly sensitive detection.

Five different solvents were selected for extractables analysis using HPLC-fluorescence. Both Millex Samplicity® 0.45 µm and 0.20 µm PTFE filters exhibited very low levels of extractables when using fluorescence detection following HPLC analysis (Figures 4 and 5).

 

  Water Acetonitrile Methanol
LC-MS-certified

syringe filter

2x106 2 x105 2.5 x107
Millex Samplicity®
0.45 µm PTFE
3 x106 3 x105 1 x106
Millex Samplicity®

0.20 µm PTFE

2.5 x106 1 x105 5 x105

Table 3. Maximum observed total ion current based on LC-MS extractables profile. The most extractables were seen with LC-MS-certified syringe filters (highlighted box).

Average total ion current for methanol extractables showing very high level of extractables from LC-MS certified syringe filters. In comparison, both the Millex Samplicity® 0.45 µm and 0.20 µm PTFE show very low levels of LC-MS extractables, providing cleaner samples for downstream analysis.

Figure 3.

Average total ion current for methanol extractables showing very high level of extractables from LC-MS certified syringe filters. In comparison, both the Millex Samplicity® 0.45 µm and 0.20 µm PTFE show very low levels of LC-MS extractables, providing cleaner samples for downstream analysis.

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Figure 4.

Fluorescence intensity with water as extraction solvent.

Fluorescence intensity using acetone as extraction solvent.

Figure 5.

Fluorescence intensity using acetone as extraction solvent.

Additional fluorescence data were also generated using methanol, acetonitrile and THF as extraction solvents, and these solvents also showed very low levels of extractables as observed by HPLC-fluorescence detection.

Conclusion

As HPLC and its downstream detectors become more and more sensitive, a greater onus falls on the sample preparation devices to provide clean extracts for HPLC. Any device that comes in contact with an HPLC sample has a potential to introduce extractable impurities into the sample.

The Samplicity® filtration system was specifically developed for filtration of samples directly into a HPLC vial, thereby avoiding filtration into tubes and transfers into vials, both of which have potential for introducing extractable impurities into the sample. Therefore, as long as the Milex Samplicity® filters themselves do not introduce any extractables, the sample should remain free of extractable impurities.

In this study, we have shown extractables profiles of Millex Samplicity® 0.45 µm and 0.20 µm PTFE filters using three different commonly used HPLC detectors. In case of UV and fluorescence detectors, both the Millex Samplicity® 0.45 µm and 0.20 µm PTFE filters provided very clean extracts without addition of extractable impurities to the samples. When using MS as the detection method, the Millex Samplicity® filters actually provided cleaner extracts than those prepared using a non-EMD Millipore LC-MS-certified syringe filter. Clearly, Samplicity® systems with Millex Samplicity® filters provide an ideal solution for preparing samples for ultrasensitive chromatographic analyses.

Materials