Testing biological tissue for abortion drugs
Photo Credit Chromatograph on Unsplash
Two studies explain proof of concept for testing for the presence of abortion drugs after a pregnancy loss using a highly specific process: ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole mass spectrometry (QqQ-MS/MS) (coupled as UHPLC-QqQ-MS/MS).
Here, I will walk through what exactly this testing process is and how it works. Then I will explain how they got the samples to do the testing and what their testing revealed; and finally, I will do some hypothesizing on the effects this research may have on abortion laws.
First things first: nerd stuff.
What are the testing processes?
Chromatography
In the context being used in these papers, chromatography is a lab technique that involves the separation of the components of a mixture, which can be a liquid, gas, or supercritical fluid. Chromatography involves 2 phases: a mobile phase and a stationary phase. The mobile phase contains the sample, the mixture, and it moves in a specific direction along or through the stationary phase. The stationary phase is the part of the apparatus or instrument that does not move, but assists in separating the mobile phase into its component parts based on specific characteristics.
Liquid chromatography (LC)
Sounds just like what the term describes! We are separating out the components of a mixture and the mobile phase is liquid, as opposed to gas or a supercritical fluid. So all the mixture components are in this liquid mobile phase. The stationary phase is a solid material and it can be packed into a column or it can be a solid plane of an object (like a piece of chromatography paper).
For example, if the mixture contains some positively and negatively charged particles, we can separate them by pouring the mixture over a glass column packed tightly with negatively charged balls of material. As the mobile phase of the mixture trickles through the solid, stationary phase of the column, the negatively charged particles in the mixture will move through more quickly and come out at the bottom of the column faster than the positively charged particles. Negative charges repel, and the negatively charged particles in the mobile phase move through the negatively charged stationary phase material without interacting. However, the positively charged particles in the mobile phase will interact with the negatively charge stationary phase, so they will be retained longer, or hang out in the glass column longer and they will come out of the bottom of the column more slowly compared to the negatively charged particles in the mobile phase.
The stationary phase can separate the components of the liquid mobile phase based on charge/polarity (as explained above) of the components, size of the components, or chemical affinities (likeliness to bind) of the components for the stationary phase.
High-performance liquid chromatography (HPLC)
It is LC taken to the next level! This technique for separating components delivers the mobile phase mixture to the stationary phase column for separation at a steady flow rate and pressure to maximize the separation of the components. HPLC instruments have a pump and other parts to deliver the mixture to the column. Usually, there is a detector at the other end of the column that detects the components as they come out and spits out a graph called a chromatogram, which tracks both the signal intensity from the component and the time it took for the component to come out. Using signal intensity information, usually an amount or a comparative amount of the components can be calculated.
In the two cases we will see about testing for abortion drugs after a fetal death, the detector is actually a separate, second instrument: a triple quadrupole mass spectrometer (QqQ MS)
Mass spectrometry (MS)
This measurement technique has 3 steps: forming ions of the components you are testing, sorting the ions based on mass-to-charge ratio, then detecting the ions and displaying the results on a chart.
An ion is a particle, whether an atom or a whole molecule, that is charged. You can have positively charged particles (cations) or negatively charged particles (anions). A mass-to-charge ratio is the mass of a molecule (in a specific set of units) divided by the total positive or negative charge on the molecule.
Most of the time, components in a mixture are not already ions; they are neutral molecules and they have to be ionized, usually by applying heat, electricity, and air to strip electrons away from the molecules and leave them positively charged. This process often breaks the molecules apart into 2 or more pieces, so part of this technique involves piecing a molecule back together from the information on the smaller parts detected.
To sort the molecules that are now cations, the cations pass through to a chamber with a magnetic field that will deflect the ions as they come through, and make them hit a detector. The heavier the cation, the less deflection — heavier cations of the same amount of positive charge will travel farther before hitting the detector than the lighter cations. However, the higher the charge, the greater the deflection. So with two cations of the same mass, the one with more positive charge will be deflected faster and hit the detector soon than the one with less positive charge.
The deflection information gives us what we need to calculate the mass-to-charge ratio, or m/z, of each molecule or part of a molecule that was in the mixture.
Triple quadrupole mass spectrometry (MS/MS or LC/MS/MS)
This technique builds on what a mass spectrometer already does!
A quadrupole is a set of 4 metal poles. The opposite poles in the setup have a voltage applied to them, and the other pair of poles may have a voltage applied to them (which may be different); this generates an electric field within the quadrupole.
Based on the electric field that has been generated, only some of the ions, called precursor ions, can travel all the way down the central axis of the quadrupole into a collision chamber which may or may not have a second quadrupole set up. The ion selected collides with neutral molecules in the chamber and fragments, and these fragments travel through to the third and final quadrupole, which is set up like the first one. Based on the applied voltage to the pairs of poles, only specific fragments of the precursor ions will travel down the central axis to the detector for the m/z ratio to be calculated.
This technique has several advantages when working with complex samples, such as very high specificity. When working with samples with many components, such as a biological tissue sample, being able to select the precursor ion based on the m/z ratio it should have is a definite advantage. Being able to tell if the molecule we are really looking for is present vs. just knowing a molecule of the same size as the one we are looking for is also an advantage — this is done with the collision and third quadrupole. Even molecules of the same mass and charge (and therefore m/z ratio) will fragment differently and therefore give different results for the m/z ratios of the fragmented parts that reached the third quadrupole.
How does it all work together?
First a biological sample must be taken. In these cases, the samples were either maternal blood, placental tissue (ground up), or fetal liver tissue (ground up). The samples were prepared specifically to allow them to go through the instrumentation and be ionized.
The sample must be injected into the chromatography part of the instrument. In each case, the researchers already knew which molecules, ions, and precursor ions they were looking for, which allowed them to choose the stationary phase and the triple quadrupole electric field that would specifically detect those molecules and ions.
How did they get the samples?
For the mifepristone study, there was one case, and they took one sample. They got a blood sample from a woman who came to a hospital with a dead fetus, a little girl about 20-21 weeks old who was likely birthed live or who was alive during the active labor part of the abortion and died shortly thereafter. The woman claimed miscarriage and later revealed she had bought abortion pills off the internet. Her blood was collected about 12 hours after the delivery and 24 hours after taking the mifepristone.
For the misoprostol study, there were two cases, and four total samples. In the first case, the baby girl was 21 weeks old when she was aborted. The mother’s blood was collected 2 days after taking the last dose of abortion pills, which she bought online. Samples of the placenta and of the fetal liver were taken 3 days after the last dose of pills. It was noted this baby could have been born alive before dying shortly after. In the second case, “On the sidewalk next to the garbage container a male human fetus was revealed (3-4 months of pregnancy), covered with clothes.” A sample of his placenta was taken 3 days after his body was found.
What did the test results reveal?
Misoprostol
Misoprostol is not discoverable in maternal blood two days after taking the pills. It is likely because it is broken down much more rapidly than mifepristone and its main metabolite (a component the body breaks misoprostol into as it metabolizes the drug) is quickly secreted by the body.
However, misoprostol can be detected in the placental tissue and the fetal liver tissue.
Toxicological analysis of the mother’s blood did not reveal any abortifacients. Misoprostol acid was found in other biological specimens [placenta] … as well as in fetal liver.
Szpot et al, “Forensic Toxicological Aspects of Misoprostol use in Pharmacological Abortions,” Molecules 27(19):6534, Oct 2022
Mifepristone
Mifepristone and its metabolites can be detected in maternal blood at least up to 24 hours after ingesting the drug.
Analysis of the maternal blood sample revealed the presence of five compounds: mifepristone (557.4 ng/mL), N-desmethyl-mifepristone (638.7 ng/mL), 22-OH-mifepristone (176.9 ng/mL), N,N-didesmethyl-mifepristone (144.5 ng/mL) and N-desmethyl-hydroxy-mifepristone (qualitatively).
Quintas et al, “Determination of Mifepristone (RU-486) and Its Metabolites in Maternal Blood Sample after Pharmacological Abortion,” Molecules 27(21): 7605, Nov 2022
The limit of quantification (the smallest amount of drug detectable above background noise) is 0.5 nanograms of mifepristone/1 milliliter of blood.
The authors were also able to use their data to propose and support a pathway of metabolism for mifepristone by the human body that had previously only been hypothesized using human blood cell experiments in a lab setting, not humans actually taking the drugs and the testing of their blood after.
What do these findings mean for the future of abortion laws?
Up until now, abortion proponents have repeatedly emphasized that there is no way to distinguish between a miscarriage and abortion via pills. These pilot studies suggest otherwise.
It’s unlikely this research will cause major changes any time soon, however. First, there are issues of resources and costs. These methods of investigation are comparably intensive. Scientists can’t use a benchtop instrument or gather the samples with a quick swab. They would have to gather blood or tissue (more involved) and send samples to a lab with more expensive instrumentation.
Second, at least in the United States, laws restricting or banning abortion focus on abortion providers and exempt women seeking abortion from prosecution. The public, including many people who describe themselves as pro-life, has little-to-no appetite for legal repercussions against women who abort. (Post-publication edit: Although there are other uses for such testing, such as identifying abortion pill providers or giving recourse to women who suspect they’ve been given abortion pills without knowledge or consent.)
Nevertheless, if the above factors were to change, there is now at least one plausible method for detecting use of mifepristone and misoprostol.
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