50 Years of TLC-MS:
Thin-layer Chromatography Coupled to Mass Spectrometry and New Perspectives by Complementary Use to HPLC as Demonstrated in Testing of Honey

Michael Schulz, Head of Instrumental Analytics R&D; Michaela Oberle, Scientist, Instrumental Analytics R&D; Markus Burholt, Scientist Instrumental Analytics R&D;
Anita Piper, Scientist Instrumental Analytics R&D; Monika Bäumle, Global Product Manager Thin Layer Chromatography

Thin-Layer Chromatography coupled to Mass Spectrometry testing of honeyThin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) are known to be convenient, fast and efficient separation techniques enabling analytical methods without the need for complicated sample preparation or high investments. Low cost and short analysis time per sample is given by parallel analysis of many samples on one plate. The high matrix tolerance of TLC offers additional opportunities to existing routine methods, such as cross-checking of HPLC results or complementary method development.

Various different detection approaches such as analyte visualization by application of derivatization reagents or coupling to other methods like UV detection can be used in combination with TLC. In 1969, Prof. R.E. Kaiser has reported the coupling of TLC with mass spectrometry (MS) for the first time.1 TLC spots were heated and desorbed into a gas stream in front of the inlet of a mass spectrometer. Numerous publications have demonstrated convincing results and contribute strongly to the progress of TLC, today and in the future.2

High-performance liquid chromatography (HPLC) is an established analytical technique for quick and highly efficient analyses of a broad range of complex samples. In contrast to TLC, HPLC can suffer from matrix rich samples causing problems such as increased backpressure or column clogging by accumulation of matrix compounds at the column inlet. In addition, the detection of ghost peaks is possible during repeated sample injections under unsuitable gradient conditions.

The joint use of TLC and HPLC is an option to combine the best out of two chromatographic worlds: High matrix tolerance of TLC makes sample preparation facile or even obsolete and HPLC provides excellent peak capacity for the efficient separation of overlapping TLC bands and increases sensitivity, compared to TLC- MS, by band focusing. Combining 2 different phase selectivities can make the TLC-HPLC-MS hyphenation a true 2D-LC method.

In this article we describe the coupling of thin layer chromatography to mass spectrometry (TLC-MS) and the combination of TLC-MS with high performance liquid chromatography (TLC-HPLC-MS) using as an example the detection of neonicotinoid pesticides in honey.

TLC-MS coupling techniques

The techniques for coupling TLC directly with mass spectrometry can be divided into elution- and desorption-based techniques.2

The elution-based approach utilizes a TLC-MS interface that enables the dissolution of the analyte from the silica plate by a solvent and transfer to the mass spectrometer in the liquid phase (see Figure 1).

 

Figure 1. Schematic working principle of elution-based TLC-MS.

Schematic working principle of elution-based TLC-MS

 

Desorption-based techniques make use of vaporization of the analyte from the TLC surface and transfer to the MS in the gas phase. Vaporization techniques include, gas beam, ion bombardment and MALDI (matrix assisted laser desorption/ionisation) or DART (direanalysis in real time).

Both approaches work offline, and both are performed after a TLC separation is finished and the plate is dried. The sample transfer to the MS is fast and typically takes less than a minute.

Features and benefits of TLC-MS

  • Sample preparation mainly takes place on the TLC plate
  • Direct MS analysis of spots or bands of interest - rapid results
  • Chromatography is performed separately from MS infusion - high flexibility in choosing mobile
  • MS-grade plates allow for high combined with high sensitivity and reliability in MS detection

Combining TLC-MS and HPLC-MS

A flexible instrument setup allows for direct elution- based TLC-MS and TLC-HPLC-MS measurements (see Figure 2). A schematic overview over the entire workflows is displayed in Figure 3. A spot can be eluted from the plate and transferred to a HPLC column for detailed analysis. Here the TLC can act either as sample preparation or as the first dimension of 2D-LC.

Features and benefits of TLC-HPLC-MS

  • High matrix tolerance of TLC allows for analyses without complex sample preparation
  • Screening and method development capabilities by parallel sample application on one TLC plate and by the option to apply a high variety of staining reagents for visual spot determination during the method development
  • Bands overlapping (not resolved compounds) on the TLC plate can be separated by the high separation power of HPLC
  • Increased sensitivity by TLC-HPLC-MS compared to TLC-MS

 

Figure 2. Schematic setup of TLC-HPLC-MS.

Schematic setup of TLC-HPLC-MS

 

Figure 3. Overview of TLC-MS and TLC-HPLC-MS workflows including instruments and consumables.

Overview of TLC-MS and TLC-HPLC-MS workflows

 

Neonicotinoids in Honey

The highly effective group of neonicotinoid pesticides is under discussion regarding negative effects on bee health. (EU) No. 485/2013 prohibits the use and sale of seeds treated with plant protection products containing the neonicotionoids clothianidin, imidacloprid and thiamethoxam. In April 2018 the EU banned these compounds on all outdoor uses (EU) 2018/783-785. European Union maximum residue levels (MRLs) of neonicotionoids authorized in food and feed products are 50 ng/g for acetamiprid, imidacloprid and thiacloprid and 10 ng/g for clothianidin and thiamethoxam.3

Honey is a product of natural origin and it is one of the most frequently tested food products. Because of its high viscosity and high sugar content, honey represents a very complex matrix.

Experimental

All TLC analyses were performed utilizing HPTLC Silica gel 60 F254 MS-grade plates.

Neonicotinoid standard solutions (NSS) 1 and 2 were prepared by dissolving 0.2 mg/mL and 1 ng/mL, respectively, of each of the seven pesticides nitenpyram, dinotefuran, thiamethoxam, clothianidin, imidacloprid, acetamiprid and thiacloprid in acetone.

Sample preparation was done by diluting 1 g honey in 10 mL water/acetone 1/1 (v/v). The samples were applied bandwise (2.5 mm band width) using a CAMAG ATS4.

The thin layer chromatogram development was performed in two steps, using acetonitrile and acetonitrile/methanol 3/1 (v/v) as mobile phases. The development time was 1 and 3 minutes. Table 2 displays an overview over all applied tracks and obtained hRf values for the analytes.

TLC-MS and TLC-HPLC-MS experiments were performed by an elution-based approach, using the CAMAG TLC- MS Interface 2 combined with a Waters Acquity® UPLC H-Class Bio System with an ACQUITY® QDa detector.

 

Table 1. HPLC conditions.

HPLC Column Purospher® STAR RP-18 endcapped (2µm) Hibar® HR 100-2.1 (Cat.No. 1.50648)
Mobile Phases [A] Water w/ 0.1 % formic acid
  [B] Acetonitrile w/ 0.1% formic acid.
Gradient 100 to 90 % A in 3 min,
  90 to 70 % A in 2 min,
  70 to 60 % A in 7 min,
  60 to 100 % A in 0.4 min,
  100 % A for 3.2 min
Flow Rate 0.25 mL/min
Column Temp Room temperature
MS Mode ESI (+)
TLC spot elution 100 % water, flow rate 0.25 mL/min

Results and discussion

Analysis of neonicotinoids in honey

In total, 33 tracks of five different samples were applied onto the TLC plate:

  • a) NSS 1 with a pesticide concentration of 0.2 mg/mL of each neonicotinoid
  • b) NSS 2 with a pesticide concentration of 1 ng/mL of each neonicotinoid
  • c) honey sample spiked with 1 mg/g of each neonicotinoid
  • d) honey sample spiked with 10 ng/g of each neonicotinoid
  • e) unspiked honey sample

Table 2 displays an overview over all applied tracks and obtained hRf values for the analytes.

Figure 4A shows the developed TLC plate under irradiation with UV light (254 nm). The neonicotinoids in spiked honey samples are visible at hRf = 70 (nitenpyram) and hRf = 93 (dinotefuran, thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid). In order to visualize the high matrix load, the plate was stained with anisaldehyde sulfuric acid reagent (Figure 4B). The long, dark smearing zone can be attributed to the high sugar content of the sample. In addition, ninhydrin staining was applied in order to visualize compounds bearing aminofunctions (Figure 4C).

After TLC development, elution-based TLC-MS was used to elute the zone at hRf = 70 and identify it as nitenpyram by subsequent single-quad MS detection. (Figure 6A).

 

Table 2. TLC data: Track numbers with applied samples and volumes and obtained hRf values were:
70: Nitenpyram; 93: Dinotefuran, Thiamethoxam, Clothianidin, Imidacloprid, Acetamiprid, Thiacloprid

Track Substance Application Volume (µL)
1, 12, 23 Neonicotinoid standard solution 1 – each 0.2 mg/mL 0.5 µL
3, 14, 25
4, 15, 26
5, 16 ,27
Neonicotinoid standard solution 2 – each 1 ng/mL 0.8 µL
1.0 µL
1.2 µL
2, 13, 24 Honey sample – spiked with 1 mg/g of each neonicotinoid 1.0 µL
9, 20, 31
10, 21, 32
11, 22, 33
Honey sample – spiked with 10 ng/g of each neonicotinoid 1.0 µL
6, 17, 28
7, 18, 29
8, 19, 30
Honey sample – without spiking 1.0 µL

 

Figure 4. A) Visualization of the neonicotinoids under UV light (254 nm). B) Visualization of matrix compounds after staining with anisaldehyde sulfuric acid (white light). C) Visualization of amino group containing matrix compounds by ninhydrin staining (white light).

Visualization of the neonicotinoids under UV light

 

The zone at hRf = 93, resulting from the TLC separation of spiked honey samples and consisting out of six analytes, was eluted from the plate onto the HPLC column. Chromatograms were obtained using UV detection for the two spiked and one unspiked honey sample (Figure 5 and Table 3). In addition, MS detection was utilized to identify the six neonicotinoids (see spectra in Figure 6 B–G).

 

Table 3. HPLC retention times of neonicotionoids in spiked honey samples after spot elution and HPLC analysis of the TLC band at hRf = 93.

Peak Substance Retention Time [min]
1 Dinotefuran   6.6
2 Thiamethoxam   7.6
3 Clothianidin   8.1
4 Imadacloprid   8.4
5 Acetamiprid   8.8
6 Thiacloprid 10.1

 

Figure 5. HPLC chromatograms of spiked and unspiked honey samples after spot elution and HPLC analysis of the TLC band at hRf = 93. A: Honey sample spiked with 1 mg/g. B: Honey sample spiked with 10 ng/g, C: unspiked honey sample. Peak IDs:1: Dinotefuran, 2: thiamethoxam, 3: clothianidin, 4: imidacloprid, 5: acetamiprid, 6: thiacloprid.

HPLC chromatograms of spiked and unspiked honey samples

 

The TLC-HPLC-MS setup was capable of detecting all neonicotinoids in the honey sample spiked at a level of 10 ng/g. As reproducibly (multiple TLC tracks) demonstrated by means of MS, the unspiked honey contained acetamiprid and thiacloprid at levels below the EU limits (MRLs) of 50 ng/g.

For precise quantification by this approach further studies are needed.

The versatile TLC-HPLC-MS setup with its TLC strengths of high matrix tolerance, high sample capacity and derivatization flexibility in combination with the high separation power of HPLC enables new approaches especially for the analysis of matrix rich and complex samples.

 

Figure 6. Analysis of honey spiked with pesticides. Mass spectra of seven neonicotinoids obtained by analysis of the TLC band at hRf = 70 using TLC-MS (A: nitenpyram) and by analysis of the TLC band at hRf = 93 using TLC-HPLC-MS
(B: dinotefuran; C: thiamethoxam; D: clothianidin; E: imidacloprid; F: acetamiprid; G: thiacloprid).

Analysis of honey spiked with pesticides - nitenpyram

Analysis of honey spiked with pesticides - dinotefuran

Analysis of honey spiked with pesticides - thiamethoxam

Analysis of honey spiked with pesticides - clothianidin

Analysis of honey spiked with pesticides - imidacloprid

Analysis of honey spiked with pesticides - acetamiprid

Analysis of honey spiked with pesticides - thiacloprid

Conclusion

An example for the analysis of different analytes in a complex and challenging food matrix was described by using TLC-MS and TLC-HPLC-MS as attractive and flexible methods. Target analytes can easily be separated and detected without time-consuming and labor-intensive sample preparation.

The flexible instrument setup enables the combination of elution-based TLC-MS and TLC-HPLC-MS measurements as complementary chromatographic methods in one setup. The applicability of this combination was demonstrated by means of the analysis of 7 neonicotinoid pesticides. Spiked and unspiked honey samples were analyzed. In the unspiked honey sample acetamiprid and thiacloprid were found at levels below the EU limit (MRLs) of 50 ng/g.

Screening and method development capabilities were shown by the application of 33 tracks (21 honey samples and 11 standard solutions). The high matrix load of the honey samples was visualized by staining with anisaldehyde sulfuric acid and the opportunity to obtain additional selective information was demonstrated by ninhydrin staining for amino group containing compounds.

The versatile TLC-HPLC-MS setup with its TLC strengths of high matrix tolerance, high sample capacity and derivatization flexibility in combination with the high separation power of HPLC enables new approaches especially for the analysis of matrix rich and complex samples.

References

  1. Chem. Br. 5 (1969) 54. R. Kaiser
  2. Trends Anal. Chem. Vol. 29 (2010), Issue 10, 1157-1171. G. Morlock, W.Schwack
  3. Science Vol. 358 (2017), Issue 6359, 109-111. E.A.D. Mitchell, B. Mulhauser, M. Mulot, A. Mutabazi, G. Glauser, A. Aebi

 

Materials