The Role of ICP-MS in Understanding the Toxicological Link Between Lead Contamination in Cannabis and Hemp Products and Human Disease – Part 2

Lead represents the most serious toxicological threat of all the heavy metal contaminants on the growing of cannabis and hemp. There is no question that our historical dependence on using materials made from lead, including car batteries, paint pigments, gasoline, plumbing, ammunition, cable sheathing, lead crystal glass, radiation protection, and solders has contributed to this problem. Decades of using these lead-based products are still having an impact on our soil and aquatic ecosystems and thus having a negative effect on the growing of cannabis and hemp. As a result, lead is getting the most scrutiny by state regulators, not only because of its historical importance as a human toxicant, but also because elevated levels of lead are being reported by researchers in many cannabis consumer products.

The first part of this two-part feature focused on the link between lead toxicity and human disease and how anthropogenic sources of lead have contributed to the contamination of cannabis and hemp grown outdoors in the US. This second part looks at the role of ICP-MS and how its development and performance improvements have been a major factor in reducing blood-lead detection levels to improve our understanding of its toxicity impact on human health.

Routine monitoring of lead

There is no question that the routine monitoring of lead has had a huge impact in reducing the number of children with elevated blood levels. Lead assays were initially carried out using the dithizone colorimetric method, which was sensitive enough, but very slow and labor intensive. It became a little more automated when anodic stripping voltammetry was developed (1), but blood-lead analysis was not considered a truly routine method until AS techniques became available. Let us take a more detailed look at how improvements in atomic spectroscopy instrumentation detection capability have helped to lower the number of children with elevated blood lead levels, since atomic absorption was first commercialized in the early 1960s.

Flame AA

When FAA was first developed, the elevated level was 60 µg/dL. Even though this is equivalent to 600 parts per billion (ppb) of lead, which was well above the detection limit of ~20 ppb at the time, it struggled to meet this level when preparation and dilution of the blood samples was taken into consideration. This typically involved either dilution with a weak acid followed by centrifuging/filtering; or acid digestion followed by dilution and centrifuging/filtering. More recently dilution with a strong base like tetramethylammonium hydroxide (TMAH) and the addition of a surfactant to allow for easier aspiration has been used. When sample preparation was factored into the equation, the concentration of lead was reduced to 10-20 ppb – virtually the same as the FAA instrumental detection limit.

Delves cup AA

To get around this limitation, an accessory called the Delves Cup was developed in the late 1960s to improve the detection limit of FAA (2). The Delves Cup approach uses a metal crucible or boat usually made from nickel or tantalum, which was positioned over the flame. The sample, typically 0.1-1.0 mL is pipetted into the cup, where the heated sample vapor is passed into a quartz tube, which was also heated by the flame. The ground sate atoms generated from the heated vapor are concentrated in the tube and therefore resident in the optical path for a longer period of time, resulting in much higher sensitivity and about 100x lower detection limits. The Delves Cup became the standard method for carrying out blood lead determinations for many years, because of its relative simplicity and low cost of operation.

Unfortunately, the Delves Cup approach was found to be very operator dependent, not very reproducible (because of manual pipetting) and required calibration with blood matrix standards (3). It was still widely used but became less attractive with the commercialization of electrothermal atomization in the early-1970s. This new approach offered a detection capability for lead of ~ 0.1 ppb – approximately 200x better than FAA. However, its major benefit for the analysis of blood samples was the ability to dilute and inject the sample automatically into the graphite tube with very little off-line sample preparation. In addition, because the majority of the matrix components were “driven-off” prior to atomization at ~ 3000 °C, interferences were generally less than the Delves Cup, which only reached the temperature of the air/acetylene flame at ~2000 °C. This breakthrough meant that blood lead determinations, even at extremely low levels, could now be carried out in an automated fashion with relative ease.

Zeeman correction GFAA

The next major milestone in AA was the development of Zeeman background correction (ZBGC) in 1981, which compensated for non-specific absorption and structured background produced by complex biological matrices, like blood and urine (4). This, in conjunction with the STPF (stabilized temperature platform furnace) concept, allowed for virtually interference-free graphite furnace analysis of blood samples, using aqueous calibrations (5). Such was the success of the ZBGC/STPF approach, due primarily to the fact that it could analyze many different kinds of samples using simple aqueous standards, that it became the recognized way of analyzing most types of complex matrices by GFAA.

Even though GFAA had been the accepted way of doing blood lead determinations for over 15 years, the commercialization of quadrupole-based ICP-MS in 1983 gave analysts a tool that was not only 100x more sensitive but suffered from less severe matrix-induced interferences than GFFA. In addition, ICP-MS offered multielement capability and much higher sample throughput. These features made ICP-MS very attractive to the clinical community, such that many labs converted to ICP-MS as their main technique for trace element analysis. Then as the technique matured, utilizing advanced mass separation devices, performance enhancing tools, powerful interference reduction techniques and more flexible sampling accessories, detection limits in real-word samples improved dramatically for some elements. Figure 2 shows the improvement in detection capability (in ppb) of ICP-MS compared to ETA and the other AS techniques.

It should also be emphasized these are instrument detection limits (IDLs), which are based on simplistic calculations of aqueous blanks carried out by manufacturers and not realistic method detection limit (MDL) or procedural limits of detection (PLOD) taking into consideration the sample preparation procedure, dilution steps and multiple analytical measurements (6). They are also only intended to be used as a guideline for comparison purposes because there are so many different ways of assessing detection capability, based on variations in manufacturer, instrument design and methodology.

Figure 2: Comparison of detection capability of AS techniques (ppb) used to monitor blood lead and the approximate year they were developed/improved.

Method/procedural limits of detection

Figure 3 is a combination of figures 1 and 2 and shows improvement in the blood lead method detection limit (now in µg/dL and not ppb) offered by the AS technique compared to the trend in blood lead levels set by the CDC. To make the comparison more valid, a factor of 100x has been applied to the instrumental detection limits to give an approximation of the achievable “real-world” method detection limit (MDL) in a blood sample matrix. Both plots are shown in log scale, so they can be viewed on the same graph. The main purpose of these data is to show the trend in elevated blood levels over the past 50 years as method detection limits of the different AS techniques have been lowered, which has given researchers more confidence in the integrity of their data.

Figure 3: The improvement in real-world method detection capability (in µg/dL) offered by AS techniques for blood-lead determinations compared to the trend in regulatory blood-lead levels.

It should also be emphasized that degradation factor of 50-100x is quite normal to convert an IDL to an MDL, when characterizing samples by AS techniques. However, when analyzing a very complex biological matrix like blood by ICP-MS, there are many different ways of calculating LODs to encompass the entire analytical procedure (often called the procedural LOD). One common approach is to carry out 20 runs and plotting standard deviation of the standards and spiked matrix versus concentration, extrapolating the regression line to the ordinate axis which will give you the standard deviation at zero concentration. In a high throughput lab, this might not be realistic, because of the additional time taken. It can be somewhat alleviated by taking less readings, but this will clearly negatively impact the statistical data and detection limit. Whichever approach is used, they should take into account variability in sample preparation, environmental contamination, solvents/reagents, as well as minor sampling errors from diluters or pipets over many runs, which can cause variability from day to day. For that reason, a real-world procedural LOD for Pb in blood is often 3 orders of magnitude worse than the instrument detection limit and is typically around 0.01-0.05 µg/dL (0.1-0.5 ppb), depending on the type ICP-MS technology and interference reduction technique used (7).

This real-world detection capability is an important distinction to make because the regulated levels for lead in cannabis and cannabis consumer products in most US states is 0.5 µg/g parts per million (ppm). When sample preparation is carried out by digesting 0.5 g of sample and diluting to 50 mL, that is equivalent to 5 µg/L (ppb) in solution. The limit of quantitation for lead by ICP-MS is around 0.0005 ppb. So, three orders of magnitude lower would 0.5 ppb, which is only 10x higher than the regulated limit for lead. The point being that only ICP-MS of all the AS techniques, would be capable of carrying out real- world quantitation at this level.

Identifying sources of lead using isotopic fingerprinting

An added benefit of the ICP-MS technique is that it also offers isotopic measurement capability, which are beyond the realms of other AS techniques. In fact, this capability allows researchers to get a better understanding of the source of lead poisoning, by measuring the isotope ratio of blood-lead samples and comparing them with possible sources of lead contamination (8). The principal behind this approach, known as isotopic fingerprinting, is based on the fact that lead is composed of four naturally occurring isotopes - ²⁰⁴Pb, ²⁰⁶Pb, ²⁰⁷Pb and ²⁰⁸Pb, all with the same atomic number but with different atomic masses So when natural-occurring lead is ionized in the plasma, it generates four ions all with different atomic masses. This can be seen in Figure 4, which shows a mass spectrum of the four lead isotopes ²⁰⁴Pb, ²⁰⁶Pb, ²⁰⁷Pb and ²⁰⁸Pb, together with their relative natural abundances of 1.4 percent, 24.1 percent, 22.1 percent and 52.4 percent, respectively.

All the lead isotopes, with the exception of ²⁰⁴Pb, are products of radioactive decay of either uranium or thorium, whose abundance will vary slightly depending on the rock type and geological area. This means that in all lead-based materials and systems, ²⁰⁴Pb has essentially remained unchanged at 1.4 percent, since the earth was first formed (9). The ratios of the isotopic concentrations of ²⁰⁸Pb, ²⁰⁷Pb and ²⁰⁶Pb to that of ²⁰⁴Pb will therefore vary depending on the source of lead. This fundamental principle can then be used to match lead isotope ratios in someone’s blood to a particular environmental source of lead contamination. However, it is important to emphasize that there are well-understood limitations of this approach. For lead fingerprinting to be useful, potential sources of lead exposure must be limited in number and scope and the lead sources must be isotopically distinct, otherwise mixed isotope ratios will occur and as a result, it will be more difficult to interpret the data. So even though it could be possible to identify the source of lead two different sources – for example lead from the consumption of cannabis compared to say lead derived from a ceramic glaze. However, it would be more complicated if there were three or more sources of lead contamination to identify, such as lead derived from the cannabis plant, together with lead from the drinking water supply and/or lead from leaded gasoline.

Mexican study

A good example of using the principle of isotope ratios to pinpoint the source of lead poisoning that worked extremely well involved a study carried out on a group of people living in a small village near Mexico City (10). A number of the residents had abnormally high levels of lead in their blood, which came from one of two likely sources – the use of leaded gasoline which had contaminated the soil; and glazed ceramic pots, which were used for cooking and eating purposes. For this experiment, the lead isotope ratios were measured using an ETV sampling accessory coupled to the ICP-MS. In this device, a heated graphite tube, similar to the type used in a GFAA, is used to thermally pre-treat the sample. But instead of using the tube to produce ground state atoms, its main function is to drive off the bulk of the matrix, before the analytes are vaporized into the plasma for ionization and measurement by the mass spectrometer. The major benefit of ETV-ICP-MS for this application is that complex matrices like blood, gasoline and pottery/clay material can be analyzed with very little interference from the matrix components (11). An additional benefit with regard to taking blood samples is that typically only a 20-50 µL aliquot is required for analysis. Figure 5 represents a schematic of how the ETV-ICP-MS system works, showing the two distinct steps – pre-vaporization to drive off the matrix components and vaporization to sweep the analyte vapor into the ICP-MS for analysis.

Figure 5: Schematic of the ETV-ICP-MS system, showing the two distinct stages – pre-vaporization (a) to drive off the matrix components and vaporization (b) to sweep the analyte vapor into the ICP-MS for analysis (11).

The lead isotope ratios of ²⁰⁸Pb, ²⁰⁷Pb and ²⁰⁶Pb to that of ²⁰⁴Pb were then determined in blood samples from a group of residents using this approach and compared with the two likely sources of lead contamination from the cooking pots and the gasoline samples. Figure 6 represents a subset of data taken from the study. It shows a plot of the ²⁰⁶Pb: ²⁰⁴Pb ratio, against the ²⁰⁷Pb: ²⁰⁴Pb ratio for the blood (□), cookware (О), and gasoline (•) samples. It can be seen from this plot that the data for the blood and cookware is grouped very tightly together around the theoretical value of the ratios (known as the primeval lead value), while the gasoline data is grouped together on its own.

Based on principal component analysis of the data, it confirms that the lead isotope ratios of the blood and cooking pots are almost identical, and are very close in composition to primeval lead, with very little addition of radiogenic lead (produced from radioactive decay). On the other hand, the alkyl lead compounds used in the production of leaded gasoline are from a different source of lead and as a result generate a very different isotopic signature. These data showed very convincing evidence that the residents of this small Mexican village were getting poisoned by the glazed clay pots they were using for cooking and eating and not from contamination of the environment by leaded gasoline, as was first suspected.

Final thoughts

There is no question that developments and performance enhancements in ICP-MS instrumentation have helped us to better understand the toxicity effects of lead over the past 50 years. In particular, it has allowed us to lower the lead threshold level of 60 µL/dL in the mid-1960s to the current blood lead reference value of 5 µL/dL. More importantly, it has helped to reduce elevated blood levels of children in the US, from 26 percent in the early-mid-1990s to less than 2 percent in 2014, as well as allowing us to get a much better understanding of the environmental sources of lead contamination. In addition, it has allowed the cannabis testing community to detect lead contaminant levels down to 0.5 µg/g in cannabis and cannabis consumer products, which is the regulated limit for most states in the US where cannabis is legal. Moreover, if the required limit ever goes down which is quite likely, when federal regulators begin to scrutinize the industry, modern ICP-MS instrumentation will be able to meet the demands with relative ease, as the limit of quantitation is approximately 3 orders of magnitude lower than the current state-based regulated limits. Finally, ICP-MS using lead isotopic fingerprinting methods offers the potential to identify the source and nature of the lead contamination.

References

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