Liquid chromatography-high-resolution-mass spectrometry (LC-HR-MS) has emerged as a powerful analytical tool for large-scale small molecule screening in biological samples. The technology is capable of determining the accurate mass and corresponding molecular formula of an analyte. Following a full scan, a particular molecular ion can be selected for fragmentation in a collision cell to create fragment ions, as the second stage of mass spectrometry (MS/MS or MS2).The fragment ions can be further fragmented in the collision cell to create next-level fragment ions, as the third stage of mass spectrometry (MS/MS/MS or MS3). If the instrument allows, additional levels of fragment ions can be created, often referred to as liquid chromatography-high-resolution-multistage-mass spectrometry (LC-HR-MSn).The multiple levels of fragmentation patterns can play a key role in compound identification by matching the fragmentation patterns to a spectral library, providing superior resolving power in screening multiple compounds simultaneously in complex biological matrices. Thus, LC-HR-MSn is a powerful tool for unknown identification in clinical toxicology cases (1,2).

Natural products are chemical substances produced by living organisms, mainly plants. Many natural products have biological effects on humans and animals. In fact, poisoning caused by plants, dietary supplements, herbal and homeopathic medications accounted for over 100,000 calls to poison control centers in the United States in 2018 (3).To help resolve these clinical toxicology cases, it is valuable to establish a solution for screening toxic natural products. However, the existing large number of toxic natural products not only have complicated chemical structures but also encompass many groups of structural analogs. These facts deny the possibility of making immunoassays for screening toxic natural products. On the other hand, the superior resolving power of LC-HR-MSn in screening unknown compounds can be utilized to establish such a solution.

To demonstrate the utility of LC-HR-MSn in screening toxic natural products, an LC-HR-MS2spectral library of 113 natural products commonly implicated in poisoning was first constructed using a quadrupole-time-of-flight tandem mass spectrometer (SciexTripleTOF 5600). Then an LC-HR-MS3 spectral library of 92 natural products (a subset of the 113) was constructed using a quadrupole-linear-ion-trap-Orbitrap tandem mass spectrometer (Thermo Orbitrap ID-X Tribrid).The spectral libraries were utilized through the corresponding LC-HR-MS2andLC-HR-MS3 assays, which were established based on routine untargeted data-dependent acquisition approaches for each instrument.

The LC-HR-MS2 assay was applied to clinical toxicology cases in which toxic natural products were involved. For instance, in one case the patients ingested a significant amount of water used to soak lupini beans and presented to the emergency department with an anticholinergic toxidrome. The LC-HR-MS2 assay effectively identified the key toxic natural products in lupini beans, i.e. sparteine, lupanine, and anagyrine, which were considered the causes of the symptoms.

The LC-HR-MS3 assay can provide additional value to the LC-HR-MS2 assay because the LC-HR-MS3 library contains both MS2 and MS3 fragmentation patterns of an analyte. When comparing the two methods using natural product-spiked urine samples, among the 80 natural products identified by both assays, the LC-HR-MS3 assay showed better limit of detection (LOD) for 43 analytes while the LC-HR-MS2 assay showed better LOD for 21 analytes. In addition, the library score that reflects the confidence level of matching the analyte spectrum to the library spectrum was also compared. At analyte concentrations from 1 ng/ml to 10 ng/ml, among those identified by both assays, the LC-HR-MR3 assay showed a higher library score for 75% of the analytes. These observations indicated that there might be potential value of employing higher-degree fragmentation patterns (MS3) in analyte identification. However, due to the variation in instrumentation and software algorithms, it is difficult to draw a conclusion here. In general, both assays worked for screening toxic natural products.

On the other hand, the involvement of MS3 fragmentation patterns did increase the depth of structural information and enhances the differentiation of structural isomers in some cases. For example, matrine and lupanine are structural isomers and also analogs, therefore the MS2 spectra of the two analytes contained mostly the fragments of same m/z values. By checking the MS3 spectra of the MS2 fragment of m/z 247.180, different MS3 fragmentation patterns were identified, and the two analytes could be differentiated. The software simulation tools predicted that the MS2 fragment of m/z 247.180 represented different fragment structures from the two analytes, thus further fragmentation was able to reveal the difference.

For natural product poisoning cases, correct identification of the culprit toxins could add value to effective patient management. Therefore, LC-HR-MSn, as a powerful tool for screening toxic natural products, can be a good choice for this purpose in specialized clinical toxicology laboratories.


  1. Wu AH, Gerona R, Armenian P, French D, Petrie M, Lynch KL. Role of liquid chromatography–high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology. Clin Toxicol. 2012;50:733–42.
  2. Mbughuni MM, Jannetto PJ, Langman LJ. Mass Spectrometry Applications for Toxicology. EJIFCC. 2016;27:272–87.
  3. Gummin DD, Mowry JB, Spyker DA, Brooks DE, Beuhler MC, Rivers LJ, et al. 2018 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 36th Annual Report. Clin Toxicol. 2019;57:1220–413.