Detailed Description of Using Stable Isotopes
The following represents a general procedure for the use of isotopically labeled internal standards for the determination of the concentration of a target analyte in an unknown sample.
- A calibration curve is created using a series of standard solutions for the analyte that are spiked with the labeled internal standard and the response factor (F) is determined.
- To a known volume of the sample to be analyzed (V1) a spike of known volume (Vspike) containing a known concentration (cspike) of a labeled internal standard is added.
- The sample cleanup procedure is performed and afterward the unknown analyte remains in a volume of solvent (V2) after the cleanup procedure.
- This solution is analyzed using LC/MS/MS and the peak area from the MRM of the labeled internal standard (AIS) and the peak area of the analyte (AX) are determined.
- The concentration of the target analyte in the solution analyzed (c2) can be calculated by substituting the known and measured quantities into:
- Because the unknown was diluted during the cleanup step, the concentration of the unknown in the original sample (c1) is calculated from
Detailed Description of the Method
The analysis technique of LC/MS/MS using MRM transitions for both unlabeled and isotopically labeled compounds provides target analyte selectivity through the use of LC retention times and MRM transitions. The high signal-to-noise ratio achieved in optimizing MRM transitions allows for high sensitivity measurements to detect low concentrations of target analytes. The isotopic uniqueness of MRM transitions between target analytes and their isotopic labeled internal standards provides a means to account for losses in sample preparation techniques and ionization efficiencies due to components of the matrix that may compete for ion formation in the source. Through the use of isotopically labeled internal standards, the analyst can be assured that both the compound identification and compound quantification is of the highest degree of precision and accuracy possible.
Liquid chromatography-tandem mass spectrometry (LC/MS/MS) provides a high-degree of accuracy and precision when making both qualitative and quantitative measurements on target analytes. Tandem mass spectrometry as a post-column detector following an LC separation provides a multitude of options for characterizing and quantifying low-levels of compounds. Predominantly there are three commercially available ionization sources that can ionize a range of compounds: 1) electrospray ionization (ESI) is well suited for the ionization of large, polar molecules, 2) atmospheric pressure chemical ionization (APCI) is well suited to ionize smaller, semi-polar molecules, and 3) atmospheric pressure photoionization (APPI) is well suited to ionize non-polar molecules. The ionization mechanisms for all three sources are uniquely different and suited for ionizing the different classes of target analytes. However, all three sources will provide a soft-ionization whereby the target analyte of molecular weight M will typically protonate in positive ion mode or deprotonate in negative ion mode. Thus, ions corresponding to mass-to-charge ratios (m/z) of [M+H]+ and [M-H]- will be observed in positive and negative ion modes, respectively. The ion that is formed directly from the target analyte in the source is called the precursor ion (or historically the parent ion) and the uniqueness of the m/z value for this ion will provide some degree of selectivity in the analysis.
There are two general types of tandem configurations for mass analyzers; tandem-in-space and tandem-in-time. Tandem-in-space instruments include triple quadrupole (QqQ) mass analyzers and tandem-in-time instruments include ion-trap (IT) mass analyzers. In QqQ instruments, the first quadrupole (Q1) is set to only pass the m/z value for the precursor ion of interest and thus enhance selectivity by excluding all other m/z ions that are formed in the ion source. In the second quadrupole (q2), the precursor ion is accelerated through an inert collision gas, which fragments the precursor ion into a series of fragment ions (historically called daughter ions). The identity and intensity of the m/z ions for the fragments are dependent on the chemical structure of the analyte and can thus provide qualitative information on the chemical structure of the analyte. The third quadrupole (Q3) can either scan the fragments produced in q2 to obtain a product ion spectrum for qualitative structural interpretation or Q3 can be set to only pass one m/z fragment ion in what is termed multiple reaction monitoring (MRM). The MRM mode provides the ultimate in sensitivity and selectivity as the MRM transition corresponds to a unique Q1 precursor ion that generates a unique Q3 fragment ion for a given analyte. MRM transitions corresponding to multiple, unique Q1/Q3 ion transitions can be iteratively scanned to continuously detect target compounds eluting from the LC column. The LC retention time provides yet another degree of selectivity for compound identification and confirmation. Similar to QqQ mass analyzers, precursor and MRM scans can be performed in ion-trap mass analyzers; however, all storage and fragmentation are done in the same volume of the ion trap. Here, a single precursor ion is stored in the trap volume following in-source ionization while other ions are ejected from the trap. Then the precursor ion is fragmented in the trap and the fragments are stored to be sequentially ejected and analyzed. To execute an MRM in an ion trap, all fragments are ejected apart from the fragment to be quantified. Other scanning modes are inherent to both mass analyzer geometries (precursor ion scans, neutral loss scans, MSn); however, the use of stable-isotopic internal standards is best suited for quantification using the MRM technique. One limitation of note when using ion trap versus quadrupole mass analyzers is that there is a “one-third cutoff rule” in ion-traps. Simply stated, product ions below one-third the value of the precursor ion m/z ion cannot be observed. Through continuous infusion of a pure compound into the ion source, instrument specific parameters (source voltages, gas flows, temperatures, collision energies, collision gas pressures, etc) are optimized for a given compound to provide the best selectivity and sensitivity for identification and quantification.
Again using the example of testosterone, selectivity through retention of the isotopic incorporation can be observed in the fragments ions as well. Testosterone produces a 97 m/z fragment ion and testosterone-[13C3] produces a 100 m/z fragment ion. Thus both the MRM transition due to the testosterone (289/97 m/z) and testosterone-[13C3] (292/100 m/z) can be independently monitored. Selectivity can also be lost if the fragmented product ion does not contain the isotopically labeled atom.
Fragment ion spectra for testosterone
Fragment ion spectra for testosterone-[13C3]
Through the use of a labeled internal standard, the same degree of ionization enhancement or suppression due to the matrix will occur for both the target analyte and the labeled internal standard. This occurs because the compounds are identical with the exception of the nuclear (neutron) components of several atoms in the labeled material. Thus the instrument response for both the target analyte and the internal standard will be nearly identical. This forms the basis for quantification using internal standards. Here the concentration of the unknown target analyte X, compared to the known concentration of the internal standard spike S, is related to the ratio of the peak areas A for the analytical standard and internal standard. The response factor (F) is determined from the slope of the calibration curve.
At times, the analytical protocol calls for a “clean-up” or sample preparation step such as solid phase extraction (SPE) to remove contaminants or concentrate the analyte of interest. The labeled internal standard can be added prior to the sample preparation step as a recovery standard. In this manner, all losses due to sample preparation for the target analyte and internal standard will be identical and carried forward through the sample preparation and analytical measurement stages of the analysis. If the labeled internal standard is added after the sample preparation step, it can only account for errors that arise during the course of the analytical measurement and is termed an injection standard.
Anthony Lagalante, Ph. D.
Consultant for IsoSciences, LLC
Associate Professor of Chemistry, Villanova University