Determination of 36 Alkylphenols and Alkylphenol Ethoxylates by LC-MS/MS

• Analyte: 36 Alkylphenols (APs) and Alkylphenol ethoxylates (APEOs)
• System: MS²Vertical 9100 LC-MSMS
• Column: Supersil ODS2 (2.1 × 100 mm, 3 μm)
• Highlight: A comprehensive LC-MS/MS method with polarity switching for simultaneous determination of 36 APs and APEOs in a single run.

Alkylphenol ethoxylates (APEOs) are widely used as non-ionic surfactants in textiles. They degrade into more toxic and persistent short-chain compounds such as nonylphenol (NP) and octylphenol (OP), which are endocrine disruptors. Many countries have restricted their use (e.g., EU Directive 2003/53/EC). This method, based on GB/T 23322-2018, employs LC-MS/MS with polarity switching for the rapid and sensitive determination of 36 alkylphenols and alkylphenol ethoxylates in textile samples.

Standards

AP and APEO standards: 36 individual compounds (including NP, OP, NPEOn, OPEOn, etc.) with purity ≥ 98%.

Reagents

Methanol (HPLC grade). Acetonitrile (HPLC grade), Deionized water(18.2 MΩ·cm), Formic acid (LC-MS grade).

Other materials

Centrifuge tubes, solid-phase extraction cartridges (for cleanup), syringe filters (0.22 μm), etc.

Standard Solution Preparation

Mixed stock solution: Prepare a mixed standard solution containing all 36 compounds at appropriate concentrations in methanol.

Calibration standards: Dilute the stock solution with methanol to obtain concentrations in the range of 5–500 ng/mL (or as required).

• Cut the textile sample into small pieces (approximately 5 mm × 5 mm) and homogenize.
• Accurately weigh about 1 g (to 0.01 g) into a 50 mL centrifuge tube.
• Add 30 mL of methanol and sonicate at 70 ± 2°C for 60 ± 5 min.
• Concentrate the extract to near dryness using a rotary evaporator at ≤40°C.
• Dissolve the residue in 2 mL of methanol and filter through a 0.22 μm syringe filter.

If matrix interference occurs, further clean up the extract using a solid-phase extraction cartridge (as described in the standard) before filtration.

LC System

EClassical 3200L HPLC system equipped with a binary pump, autosampler, and column oven.

Mass Spectrometer

MS²Vertical 9100 triple quadrupole mass spectrometer with electrospray ionization (ESI) source.

Column:                      Supersil AQ-C18, 2.1×100mm, 3μm

Mobile phase: A: Water (containing 0.1% formic acid); B: Acetonitrile, in gradient (Table 1)

Flow rate:                  0.3 mL/min

Column temperature:      40°C

Injection volume:             10 μL

Table 1. Gradient program

Ionization mode:ESI positive for ethoxylates, ESI negative for alkylphenols (polarity switching)

Spray voltage: 4800 V (positive), -4500 V (negative)

Ion source temp.: 300°C

Curtain gas: 20 psi (N₂)

Nebulizer gas:           20 psi (N₂)

MRM parameters: For each compound, the precursor ion, product ions, collision energy, etc., were optimized

Typical Chromatograms

The MRM chromatograms of the 36 compounds at 50 ng/mL are shown in Figure 1, and Figure 2 displays individual MRM chromatograms.. All peaks are well-shaped and baseline separated, with sufficient data points for accurate quantification. Polarity switching allows simultaneous detection of both APEOs (ESI+) and APs (ESI-) in a single run.

Figure 1. MRM over-lay chromatograms of the 36 compounds at 50 ng/mL

Figure 1. MRM chromatograms of the 36 compounds at 50 ng/mL

Linearity and Sensitivity

Calibration curves constructed over the relevant concentration range show excellent linearity with correlation coefficients > 0.999. The method detection limits are far below the requirements of GB/T 23322-2018.

Repeatability

The method demonstrates excellent reproducibility with RSD values below 5% for most compounds.

The LC-MS/MS method using polarity switching on the EClassical 3200L and MS²Vertical 9100 system enables rapid, sensitive, and reliable determination of 36 alkylphenols and alkylphenol ethoxylates in textile samples. It provides a high-throughput screening tool that meets the stringent requirements of current regulations.