Radiolabeling compounds prior to ADME testing is just another way to make new drugs safer.

As the cost of drug development significantly increases, the importance of early evaluation of drug candidates and successful absorption, distribution, metabolism, and excretion (ADME) studies is becoming more evident. For use in ADME studies, radiolabeled compounds synthesized and tested in accordance with good manufacturing practices (GMP) enable research groups to establish better testing parameters with higher confidence in the results.

Radiolabeling and analysis methods, such as specific activity (SA) determination and analytical HPLC, performed in compliance with GMP, assure adherence to strict training, operational, reporting, storage, and production principles and allow researchers to determine precise measurements of compound identity, purity, and activity for establishing improved ADME study inputs.

Additionally, safety in radiolabeling procedures is of particular importance given the inherent hazards in handling radioactive materials and the effects of ionizing radiation, which in large enough doses, can damage living cells and cellular structures. Time, distance, and shielding are critical elements that must be controlled to ensure the protection of people working with radioactive materials that emit penetrating radiation.  Individuals handling radioactive materials should consult with a Radiation Safety Officer (RSO) or local radiation protection professional to ensure appropriate radiological handling techniques.

In PerkinElmer’s laboratories, drug candidates that are radiolabeled for use in ADME studies are synthesized and analyzed in compliance with GMP principles (ICH Q7A, Good Manufacturing Guidance for Active Pharmaceutical Ingredients, Section XIX, API’s for use in clinical trials (2001)). This article describes how those principles are applied to the determination of SA, the identity and the purity of the compounds manufactured.

Importance of specific activity determination
Specific activity (SA) is the amount of radiolabeled mass in a sample, measured in millicuries (mCi) or curies (Ci) per mass (in mg) or mmol. SA of a radiolabeled substance is distinct from its concentration—a critical difference of importance to radiolabeling that cannot be minimized. While SA is a fixed physical property, concentration describes the amount of radioactivity in a solution and varies with the amount of solution used.

ADME studies test the mass limits of toxicity and the mechanisms of metabolism. SA is important for calculating the dose requirement for a study. The higher the SA, the lower the amount of mass one can test in an ADME study.

SA can be determined by a weight assay or mass spectrometry. In this example, the SA is established by a weight assay, which is then compared to the value calculated using a mass spectrum. If the value obtained using the weight assay is significantly lower (by >5%) than the mass spectrometry value, the root cause is investigated. In this case, the sample used for the weight assay may need to be re-dried under vacuum and re-assayed.

Table 1. Example of a weight assay for SA determination.
Material	Mass of Radio-labeled Compound (mg)	Volume of Solvent for Dilution (ml)	Volume Assayed (µl)	Disintegrations Per Minute (dpm)	Average dpm	SA Calculation	SA (mCi/mmol)
Sample 1	0.625	10	a. 25	517,360	516,415	(516,415 dpm x 10 ml x 404.36 mg/mmol)/(2.22 x 109 dpm/mCi x 0.025 ml x 0.625 mg)	60.20
Sample 1			b. 25	515,469
Sample 2	0.641	10	a. 25	527,905	526,003	(526,003 dpm x 10 ml x 404.36 mg/mmol)/(2.22 x 109 dpm/mCi x 0.025 ml x 0.641 mg)	59.79
Sample 2			b. 25	524,100
Sample 3	0.398	10	a. 25	326,306	327,497	(327,497 dpm x 10 ml x 404.36 mg/mmol)/(2.22 x 109 dpm/mCi x 0.025 ml x 0.398 mg)	59.95
Sample 3			b. 25	328,688
						AVERAGE SA	59.98

 

Weight assays are performed by weighing out triplicate quantities (0.1–5 mg) of the radiolabeled compound and diluting each in the same volume of solvent. Duplicate aliquots of these dilutions (designated as “a” and “b” in Table 1) are counted for radioactivity using a liquid scintillation counter and expressed in disintegrations per minute (dpm). The total amount of radioactivity is then calculated for each sample and divided by the mass of the sample. SA (in mCi/mmol) is obtained by multiplying mCi/mg by the molecular weight. The average of the three weight assay values provides the average SA by weight assay.

click to enlarge Figure 1. Mass spectrum of a 14C compound. (Source: PerkinElmer)

Determining SA by mass spectrometry is straightforward. For each ¹⁴C atom in the molecule, the m/z (mass to charge ratio) of the parent ion is increased by two units. The peak intensities of the plus two ions are compared to the peak intensity of the non-radioactive species, as shown in Figure 1.

The equation for calculating the SA by mass spectrometry is given below:
SA of a ¹⁴C compound = (((IM+2) (1) (Y)) / (IT) + (((IM+4) (2) (Y)) / IT)+ etc.

Where
I = relative intensity of observed peaks
IT = total relative intensities = IM + (IM+2) + (IM+4) + etc.
Y = theoretical SA of 1 x ¹⁴C
M = unlabeled species
M+2 = species with 1 x ¹⁴C atom attached
M+4 = species with 2 x ¹⁴C atom attached
In this example,
M = 372, M + 2 = 374, IM = 1.63, IM + 2 = 100,
so the theoretical activity of the ¹⁴C compound is 62.4 mCi/mmol. 

The SA calculation = [(100)(1)(62.4)]/101.63 = 61.40 mCi/mmol

Table 2. SA by Weight Assay SA by Mass Spectrometry % Difference 59.98 61.40 2.4%

Because it does not factor in the weight of the samples, (the sample may not be completely dry), the mass spectrometry analysis produces a higher value for SA as shown in Table 2.

 

HPLC methods for radiochemical purity analysis
High performance liquid chromatography (HPLC) is the method of choice when performing radiochemical purity analysis of a GMP product. HPLC analysis of the radioactive compound is performed with a radiometric detector in line with the UV detector.

click to enlarge Figure 2. Detector offset.  Detector offset – (0.368 – 0.250) = 0.168 (Source: PerkinElmer)

The benefit of HPLC radiochemical purity analysis is that any labeled byproduct or impurity can be easily determined. Ratios of the radioactive signal of impurities relative to the peak of interest are directly correlated. Chemical purity values based on UV can be deceiving as impurities have different extinction coefficients. Thus, the amount of signal or percent area of the UV peak does not correlate with the percentage of impurity to desired product.

Co-Elution
As an indicator of the identity of the labeled compound, its co-elution versus an unlabeled standard is confirmed by determining sample and detector offsets. These offsets can be assessed by removing the column from the HPLC system and replacing it with a short length of 0.010″ ss tubing. A mixture of the unlabeled standard and the labeled compound are then injected. The measured difference between the peak centers is considered to be the detector offset.
 
Once the detector offset has been determined, the next step is to replace the column on the HPLC system and then calculate the sample offset, as described above. The labeled compound is said to have co-eluted with the unlabeled standard if the difference in retention between the UV and radioactivity traces coincides with the instrument offset ± 0.2 minutes. For example:
Detector offset = 0.168 min ± 0.2 = 0.0 to 0.368 min
Sample offset = 0.209 min (within variability limits)

Limit of detection
ADME studies require products that are >98% pure. The limit of detection (LOD) of the radiometric detector should be determined to understand the limits in analyzing very small impurities. Using a one-minute region of background, the height of the baseline in this region is compared to the height of the radiochemical peak. The limit of detection is calculated in our laboratory as shown in Equation 1:

Equation 1.
%LOD =	3 x baseline height	x 100
	product peak height

 

click to enlarge Figure 3. Expanded baseline region. The baseline height = 6.090 – 5.937 mV = 0.153 mV. The height of the radioactivity peak (not shown) is 511.750 mV. The %LOD = 3(0.153 mV)/(511.750 mV) x 100 = 0.09% (Source: PerkinElmer)

Mass effects
Mass effects occur when the amount of radioactive product blended with unlabeled product is very small compared to the mass of the unlabeled product. This results in the UV channel becoming saturated while the radiochemical signal is insufficient or near the limit of detection. Co-elution is determined using the retention time of the leading edge of the peak rather than the top of the peak. If the radiochemical peak becomes distorted, the determination of co-elution is compromised. An HPLC experiment using increasing dilutions of the radioactive product blended with non-labeled product shows that the peak retention times of the radioactive trace and the UV trace begin to converge with each successive dilution, proving that there is indeed co-elution. When both the radioactivity and UV peaks are on-scale, co-elution can be determined by measuring the peak offset from the center of the peaks.
Offsets in the chromatograms below must be determined based on the leading edge position.

click to enlarge Figure 4. Offset measured from the leading edge. (Source: PerkinElmer)

Recovery
Good laboratory practice includes showing that the entire radiolabeled sample is eluted from the column. Radiometric recovery detection is superior to UV recovery detection and offers two simple ways for performing the analysis.

 The offset scan can also be used to determine the recovery of radioactivity from the column. Equal volumes and concentration of radioactivity can be injected with and without the column in line. Injection volume or concentration may be adjusted to keep peaks on scale. Recovery of radioactivity is calculated as shown in Equation 2:

 

The recovery of radioactivity must be 100% ±10%

Equation 2.
Recovery =	Total Peak Area with column in line	x 100
	Total Peak Area without column in line

 

 

In lieu of a radioactivity recovery injection, late-eluting impurities can be detected by eluting the sample with 100% of strong solvent for at least two column volumes. If any radiochemical impurity is detected, the original conditions must be modified and the sample re-analyzed.

Conclusion
The analytical methods described and used in our laboratory for the determination of the specific activity, identity and purity of the compounds radiolabeled for ADME studies assure basic compliance with GMP principles and should enable research groups to establish better testing parameters with higher confidence in the results.

About the Authors
Patricia Mayer is Global Product Portfolio Leader for Research Reagents at PerkinElmer Life and Analytical Sciences, leading product development in numerous areas including NEN Radiochemicals and TSA.

Linda Lacy is Operations Director at PerkinElmer Life and Analytical Sciences, where, among other duties, she is responsible for managing production of isotopically- and non-isotopically-labeled reagents.

Mario Maniscalco is Operations Manager at PerkinElmer Life and Analytical Sciences, responsible for managing production of custom radioisotope orders.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 10, October, 2008, pp. 31-33.

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Filed Under: Drug Discovery

 

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