Antiretroviral bioanalysis methods of tissues and body biofluids

Abstract

Research in the many areas of HIV treatment, eradication and prevention has necessitated measurement of antiretroviral (ARV) concentrations in nontraditional specimen types. To determine the knowledgebase of critical details for accurate bioanalysis, a review of the literature was performed and summarized. Bioanalytical assays for 31 ARVs, including metabolites, were identified in 205 publications measuring various tissues and biofluids. 18 and 30% of tissue or biofluid methods, respectively, analyzed more than one specimen type; 35–37% of the tissue or biofluid methods quantitated more than one ARV. 20 and 76% of tissue or biofluid methods, respectively, were used for the analysis of human specimens. HPLC methods with UV detection predominated, but chronologically MS detection began to surpass. 40% of the assays provided complete intra- and inter-assay validation data, but only 9% of publications provided any stability data with even less for the prevalent ARV in treatments.

Background

Current HIV clinical research objectives

The current focus of HIV clinical pharmacology and therapeutics research is in prevention of transmission, eradication of latent HIV reservoirs, treatment of co-infection with hepatitis C virus, tuberculosis and malaria, as well as the development of nanomedicine and new formulations for dose optimization. To determine compartmental distribution of antiretrovirals (ARVs) to latent viral reservoirs, prevent viral transmission, assess adherence or long-term ARV exposure, as well as investigate the etiology of adverse ARV effects, such as mitochondrial toxicity, cognitive impairment and muscle wasting, the collection and analysis of samples other than blood and its products is required. Consequently, many research protocols that include HIV clinical pharmacology objectives are developed to include the collection, handling and bioanalysis of uncommon specimen types such as biofluids and tissues. New analyses of tissues and biofluids have been reported with correlation to clinical outcomes and minimum inhibitory/effective antiviral concentrations. ARV concentrations in hair have recently been used to correlate with plasma concentrations and provide a noninvasive tool to quantitate long-term, systemic ARV exposure for HIV treatment and adherence [1–5]. Saliva drug concentrations have also been correlated with free-drug and plasma concentrations [6,7]. Cerebrospinal fluid (CSF) concentrations have been of interest for studies focused on HIV-associated neurologic disease and to calculate the ARV central penetration effectiveness score [8]. Vaginal and cervicovaginal fluids (CVF) or semen drug concentrations are currently being determined in pre-exposure pro-phylaxis and to prevent HIV transmission [9,10]. The reported concentrations in each of these specimen types may be normalized for protein content [11], cell number [12] or cell type [13], or other physiological or chemical characteristics that assist in data interpretation and for PK and PD modeling.

Importance of the specimen integrity & identity

The proper collection, storage, processing and bioanalysis of the contents of these matrices, as well as the appropriate use of concentration results in interpreting outcomes, have not been standardized. Data published from clinical studies may or may not reference or include methods for sample handling and bioanalytical methods (herein referred to as ‘methods’). While the proper collection, handling and storage of these specimens are essential to the validity of the drug concentration results, standardized methods are currently lacking. Published ARV methods that test tissues and biofluids offer an opportunity to identify specimen handling data that may expedite current research initiatives. Pre-analytical details that support the integrity of future specimens may provide researchers with valuable information for these special specimen types.

Dependability of bioanalytical methods

Methods to quantitate drugs and metabolites in nontraditional, or ‘rare’, matrices such as tissues and biofluids are not commonly required for pharmaceutical bioanalysis and are not currently regulated by US FDA guidelines [14]. However, some publications may contain important bioanalytical details that have been employed, such as precautions and conditions for stable sample preparations, prior to chromatographic analysis and the corresponding parameters of the method operation (i.e., MS detection modes). Those that have been validated and include the supporting validation data in the published method should be considered as the most dependable and valuable to the researcher, whereas those that omit these key data should be closely examined before adopting information.

Interpreting results

Finally, the appropriate use of drug concentration results from specific specimen types should consider the biological and physicochemical qualities of specimens between and within individuals and methods to provide a meaningful normalization of drug concentrations. Validation data, such as LLOQ, variability, accuracy, specificity and selectivity, can also provide a baseline for the further development or adaptation of specific methods.

Rationale for this review

This review provides a summary of pre-analytical, analytical and postanalytical material from the literature of published methods of bioanalysis for ARV concentrations in nontraditional specimens such as tissues and biofluids.

Literature searches & data compilation

Multiple Pubmed literature searches were performed to identify published ARV methods for the following specimen types:

• ■Biofluids: amniotic, breast milk, cerebral spinal, cervical/vaginal, intestinal, saliva, semen, sputum;
• ■Tissues: brain, colemic, fetal, glandular, gastrointestinal, hair, heart, kidney, liver, lung, lymphatic, muscle, nails, placental tissue, spleen, testes and vagina.

The searches did not include blood, blood cells, plasma or urine. Keywords for the searches included the drug name plus the various tissues and fluids. All approved ARVs were cross-referenced with the specimen types. The publications included analytical methods used to characterize ARVs in humans and animals.

Extraction of data

Important variables relevant to method validation and application were extracted from the publications for support of specimen handling and analysis in future clinical studies. For each publication, the ARVs and metabolites analyzed were noted. Specimen collection, handling (including addition of stabilizers), processing steps (e.g., flash freezing or homogenization prior to storage), and storage techniques, as well as the stability data associated with these steps, were identified. Stability data included duration of stability, temperature and condition of specimen. In addition to the type of analytical method used, supporting validation data, variation, accuracy and LLOQ were collected.

Classifying the specimens

After compiling the reported data from all references, final categories of specimen types and analytical method types were utilized to consolidate and coordinate all data. The tissue categories were internal tissues, hair (the only external tissue found), placenta and fetal tissue. The biofluid categories included amniotic fluid, breast milk, CSF/extracellular cerebral fluid, CVF, saliva, and semen/seminal fluid.

Enumerating results

To simplify the enumeration process for bioanalytical details, references that were cited and used for more than one specimen type were counted as a separate method (e.g., if a reference measured an ARV in both seminal plasma and saliva, it was counted as one method for seminal plasma and one method for saliva). This approach accounts for enumerated variables that differ depending on the specimen type. In addition, for references that indicated the use of multiple methods for bioanalysis, each method noted was counted as a separate method. When ‘% of methods’ is noted, the denominator is based on the above enumerations. When ‘% published’ is noted, the percentage is derived specifically from the number of total references.

Bioanalysis methods for ARVs in tissues & biofluids

The literature search resulted in the identification of 205 publications identifying methods used for measuring ARV in the nontraditional tissues and biofluids of interest [3–7,11,15–213]. 12% of the publications found measured ARV in both tissues and biofluids of interest; 5% of publications indicated multiple methods were used. As defined above for enumeration, 35–37% of the tissue or biofluid methods quantitated more than one ARV. 18 and 30% of tissue or biofluid methods, respectively, analyzed more than one specimen type. 20 and 76% of tissue or biofluid methods, respectively, were used for the analysis of human specimens. Notably, hair, saliva and CVF methods were only used for human specimens. Methods for all currently marketed ARVs were identified for at least one specimen type with the exception of tipranavir. Other than for zidovudine phosphorylated metabolites, only one other method for nucleoside reverse transcriptase inhibitors (NRTI) phosphorylated metabolites (stavudine triphosphate) was identified [203].

Sample collection

For tissues (including internal, fetal and placental), 76 of the 84 references provided a collection method. Of these, most tissues, except hair, were surgically extracted, especially when taken from animals. Human hair was collected by manually cutting a length from the head. Exsanguination of blood from tissues was not indicated in the methods used except for fetal tissue; exsanguination is important due to the contamination of blood from the vessels in the tissue. Fluids were collected using a variety of techniques appropriate to the fluid location. With the exception of breast milk, across all categories, nearly all of the methods provided a collection description. Amniotic fluid was collected during surgical procedures such as laparotomy, during delivery or directly withdrawn through a catheter, the uterine vein or a syringe aspiration. Breast milk was collected manually for five of the 15 methods, by an electric pump for one of the 15, but nine of 15 of the breast milk methods provided no collection information. Most of the CSF specimens were collected by cisternal or lumbar puncture, the latter being preferred. The two references collecting extracellular cerebral fluid used microdialysis probes in animals. A total of 23 of 43 references for seminal fluid/semen reported using masturbation; one reported using electroejaculation. Saliva and CVF collection methods varied the most and nearly all references reported the method used for collection. Saliva was collected under stimulated (Salivette®) or unstimulated conditions (expectoration) into vessels or with swabs (Salivette swab). CVF was collected by aspiration, lavage, or Sno-Strip™ or blotting paper. Neither sample rejection criteria nor notable precautions were provided for tissues or biofluids.

Sample handling & storage

Only one of the references used a buffer addition to CVF after collection for stabilization [212]. Both tissues (23%) and biofluids (5%) used cooling by placement on ice (or refrigeration) or flash freezing in liquid nitrogen in an effort to minimize or slow any decomposition or metabolism of the analytes of interest within the specimen. 63% of the methods indicated whether the tissue was homogenized prior to or after storage. Of the 63%, most tissues (60%) were stored frozen and then were thawed and homogenized prior to analysis rather than before storage (40%). 79% of the tissues and 60% of the biofluids indicated storage conditions. When indicated, freezer storage was most often indicated as an ultracold freezer (30% tissues, 65% biofluids) or by a typical freezer (52% tissues, 33% biofluids). When storage for hair samples was indicated (67%), the samples were wrapped in foil to protect from light and held at ambient temperature with or without enclosure in a desiccated bag.

Bioanalytical methods

Table 1 summarizes the method types for tissues and biofluids. The early references were more likely to have used HPLC with UV detection and, conversely, the newer references were more likely to have used a MS for detection. Some ligand-binding methods (e.g., enzyme immunoassay and radioimmunoassay) were used to measure didanosine, stavudine and zidovudine. The use of UPLC was cited in more recent methods.

Table 1.

Summary of methods used to analyze antiretrovirals in biofluids and tissues^†.

Fluid type	Total number of methods	HPLC–UV (n)	HPLC–MS (n)	HPLC–MS/MS (n)	All ligand binding‡ (n)	Other assay (n)
Amniotic fluid	33	24	1	4	3	1§
Breast milk	19	9	1	5	3	0
Cervicovaginal fluid	13	4	4	4	0	1¶
CSF and ECF	85	50	6	18	9	3#
Saliva	15	6	1	5	0	1
Male seminal plasma/semen	45	21	1	18	1	4#
Total	210	114	14	54	16	10
Tissue type	Total number of methods	HPLC–UV (n)	HPLC–MS (n)	HPLC–MS/MS (n)	Radioimmunoassay (n)	Other assay (n)
Fetal	15	9	0	3	2	1§
Hair	6	2	0	4	0	0
Placenta	12	7	0	3	2	0
Internal	38	24	8	2	1	3††
Total	71	42	8	12	5	4

^†For specific references see Supplementary Table 1.

^‡Includes all ligand-binding assays (e.g., radioimmunoassay and enzyme immunoassay).

^§Capillary electrophoresis–UV.

^¶UPLC–MS/MS.

^#HPLC–fluorescence detection, UPLC–MS.

^††UPLC evaporative light scattering, fluorescence microscopy, UPLC–MS.

CSF: Cerebrospinal fluid; ECF: Extracellular cerebral fluid.

ARVs measured

The number of methods by drug and by specimen category is depicted in Figure 1 and displayed by areas of HIV research. These research areas include HIV prevention (genital tract specimens), HIV treatment and adherence (hair, saliva specimens), HIV eradication (sanctuary site specimens such as CSF and internal tissues) and HIV maternal-to-child transmission (amniotic fluid, breast milk, fetal and placental tissue). Overall, methods for CSF analysis predominated for biofluids, while internal organs were the most commonly investigated tissues. Excluding metabolites, zidovudine (n = 81 methods) was measured most often in all categories, followed by indinavir and lamivudine (n = 37), lopinavir and ritonavir (n = 32, 34), stavudine and didanosine (n = 27), nelfinavir and nevirapine (n = 24), abacavir (n = 17), and efavirenz and saquinavir (n = 15). Very few methods measured the currently favored ARVs atazanavir or tenofovir (n = 11), maraviroc or raltegravir (n = 10), darunavir (n = 4), emtricitabine (n = 3), etravirine (n = 1) and none were identified that measured tipranavir or the phosphorylated NRTI for abacavir, lamivudine, didanosine, dideoxycytidine or emtricitabine. There was a negative correlation with the chronology of drug development and the number of ARV methods published that assayed alternative specimens (data not shown).

Figure 1. Number of biofluids and tissues methods shown by areas of HIV research.

(A) Hair and saliva as antiretroviral treatment adherence specimens. (B) CSF and internal tissue as eradication specimens. (C) CVF and semen/seminal plasma as prevention specimens. (D) Fetal and placental tissues, amniotic fluid, and breast milk as maternal-to-child transmission. 3TC: Lamivudine; ABC: Abacavir; APV: Amprenavir; ATV: Atazanavir; CSF: Cerebrospinal fluid; CVF: Cervicovaginal fluid; D4T: Didanosine; DDC: Dideoxycitabine; DDI: Didanosine; DRV: Darunavir; ECF: Extracellular cerebral fluid; EFV: Efavirenz; ETR: Etravirine; FTC: Emtricitabine; IDV: Indinavir; LPV: Lopinavir; MVC: Maraviroc; NFV: Nelfinavir; NVP: Nevirapine; RAL: Raltegravir; RTV: Ritonavir; SQV: Saquinavir; TFV: Tenofovir; TPV: Tipranavir; ZDV: Zidovudine.

Assay validation

Validation data was provided for methods for 54% of the tissue methods and complete data, including variation and accuracy at three levels of concentrations, which were provided for most of the 54% (74%), although in some cases it was difficult to determine if the validation data were specific to the matrix. Internal tissue methods provided the least (26%) validation data. For biofluids, 61% of the biofluid methods included validation data; variation and accuracy at three levels of concentrations for 68% of those reporting validation data.

Stability data

Minimal stability data for the analytes in tissues or biofluids for postcollection processing, time of storage and/or handling were reported. Table 2 summarizes stability data that were published that indicated specimen types, analytes, time and conditions of stability tested. The table also cites the reference numbers. Only 9% of the references provided stability data. For the current agents of most interest, very limited or no stability data were found: atazanavir (none), darunavir (none), efavirenz (hair – 4 months ambient) [5], emtricitabine (none), lopinavir (hair – 4 months ambient) [5], maraviroc (none), raltegravir (none) and tenofovir (vaginal tissue – 2 months ultracold) [55]. For di- or tri-phosphorylated forms of NRTI, stability data for only zidovudine forms were identified [100].

Table 2.

Summary of available antiretroviral stability data in biofluid and tissue specimens.

Matrix	Drug(s)	Stability (time)	Storage temperature (°C)	Ref.
Tissues
Internal tissue: brain	Nevirapine	48 h	Ambient	[186]
Internal tissue: brain, liver	Ritonavir	1 week	−20	[19]
Internal tissue: brain, kidneys, muscle, spleen, liver, heart, lung, lymph node, thymus	Zidovudine, zidovudine phosphorylated metabolites	4 months	−20	[100]
Internal tissue: alveolar cells, tonsil tissue	Didanosine, stavudine	3 months	−80	[112]
Internal tissue: vaginal tissue	Tenofovir	2 months	−70	[55]
Hair	Lopinavir, ritonavir, efavirenz	5 days	4	[5]
		4 months	Ambient
Fetal tissue	Dideoxycytidine	22 h	Ambient	[43]
	Abacavir	11 h	Ambient	[45]
	Abacavir, zidovudine	Chromatography samples: 24 h	Ambient†	[187]
		Three freeze–thaw cycles	Freeze–thaw conditions
Placental tissue	Dideoxycytidinelamivudine	Chromatography samples: 22 h	Ambient†	[43]
		Three freeze–thaw cycles	−20
	Abacavir	11 h	Ambient	[45]
	Abacavir, zidovudine	Chromotography samples: 24 h	Ambient†	[187]
		Three freeze–thaw cycles	−20
Fluids
Amniotic fluid	Dideoxycytidine, lamivudine	22 h	Ambient	[43]
	Abacavir	11 h	Ambient	[45]
	Abacavir, zidovudine	Chromatography samples: 24 h	Ambient†	[187]
		Three freeze–thaw cycles	Freeze–thaw condition
Breast milk	Nevirapine	4 h	Ambient	[85]
		Three freeze–thaw cycles	Freeze–thaw at −80
	Lamivudine, stavudine, zidovudine, nevirapine, nelfinavir, ritonavir, lopinavir	24 h	Ambient	[197]
		72 h	4
CSF and ECF	Didanosine, stavudine	3 months	−80	[112]
	Lopinavir	Test samples: 18h	−70	[51]
		Chromatography samples: 96 h	4
	Amprenavir	1 h	60	[184]
		24 h	25
		7 days	2
		43 days	−20
	Maraviroc	6 h	Ambient (+ light)	[108]
		Three freeze–thaw cycles	−20
		Chromatography samples: 79 h	Ambient (+ light)
Saliva	Nevirapine	17 h	Ambient	[198]
		2 months	−40
	Efavirenz	48 h	4	[52]
Semen and seminal	Didanosine, stavudine	3 months	−80	[112]
plasma	Lamivudine, zidovudine	18 months	−70	[131]
	Amprenavir	1 h	60	[184]
		24 h	25
		7 days	2
		43 days	−20
	Efavirenz	6 months	−80	[88]
		Three freeze–thaw cycles	−80
		Three freeze–thaw cycles	−20

^†Assumed since no autosampler temperature was specified.

CSF: Cerebrospinal fluid; ECF: Extracellular cerebral fluid.

Reporting of results

Concentration data were reported in mass or molar units per volume of tissue homogenate or biofluid. Some tissues methods normalized homogenate results by the weight of the tissue specimen and reported per gram unit of tissue rather than volume of homogenate. One CSF method used normalized CSF concentration by the individual total CSF protein concentration, however, there was no attempt to normalize results for other biofluids, which can also vary in protein content [11].

Key discussion points

It is clear that many physiological and practical considerations should be deliberated and with the exception of the actual method, standardized methods and approaches ought to be utilized in clinical research practices. For the method of ARV measurement, even though the FDA bioanalytical method guidance requires validation only when matrices can be obtained in adequate quantities, the pivotal nature of drug concentration data within current research initiatives may outweigh the justification of FDA guidance exemptions. These points and others follow.

A good specimen is vital

The characterization of specimen collection and handling is critical to interpreting concentration results and their relationship to therapeutic outcomes. The data collected show little variability in collection techniques, except for saliva and CVF. Methods for CVF include lavage, direct aspiration and use of Sno-Strip devices (or similar products) for collection. For CVF collection methods, the use of TearFlo® or Sno-Strip collection devices have been recommended by one set of investigators to minimize contamination of the sample with tissue cells and blood, as well as standardize the location and total volume of the collection [150]. For saliva samples, stimulated collection devices or stimulated or nonstimulated expectoration were the most common collection methods, however, saliva itself can vary in viscosity. A higher level of stimulation decreases the viscosity of the sample. Therefore, stimulation may allow for a more homogenous sample representing free-drug concentration whereas an unstimulated sample may contain more cells and other debris such as mucus and glycoproteins.

A 2011 review article reported on both the female and male genital tract samples that have been collected and analyzed for ARV concentration. Although the review is focused on the postdose ARV genital track to blood plasma ratio, the authors included various types of collection (for female only; male collections were identical) and types of analysis including the LLOQ for either plasma or the matrix [214]. The article also discusses the specimen collection method, the variability of the specimens collected and the effects on the integrity and interpretability of the sample results.

Lastly, it is important to consider conditions that render the biological samples unusable. Contamination with blood is one example, but there are additional variables (other than contamination) to be considered.

Lability & stability

Maintaining the stability of drugs within specific matrices is essential to assuring accuracy in the values reported. The contents of most tissues and biofluids contain enzymes and the pH range of some may be considerable. Since the data supporting ARV stability during specimen collection was rarely reported, laboratories will need to determine the validity of adopting the specimen collection process prior to application to clinical specimens. Moreover, data to support the stability of drugs and metabolites after freeze-thaw treatment or for long-term storage of specimens were insufficient for the application to most multicenter clinical studies. This last point of discussion should also serve as a reminder that tissue and biofluid samples may be limited by volume and/or require special handling and storage conditions that are routinely different than that of blood, plasma or urine. However, this should not excuse the testing of ARV stability under the special handling and storage conditions required, even if the measure is solely qualitative.

Methods from the past to the present

Chronologically, the primary method of analysis was HPLC with UV detection followed by single or triple quadrupole MS detection in more recent years. These methods represent a foundation upon which newer, more complex technology can be built. The bioanalytical field can continue to expect less HPLC and more UPLC, which produces shorter analysis times and utilizes less solvent. Detectors will also continue to become more sensitive, specific and selective to allow for measurements below the femtomole range, in targeted tissues or biofluids, with small differences in mass or chemical properties, respectively. These detection improvements will ultimately be needed as the development of nanoformulations matures and products are approved based on tissue-targeted concentrations.

Although not reported in this literature review, quantitation of ARVs in internal tissues using radioactively labeled drugs in animals was common. Radioactively labeled compounds can be more sensitive, but are often less specific and are subject to more matrix effects. The same is true of ligand-binding methods for small molecules such as ARVs. Few references cited the use of ligand-binding methods for ARV measurement and, in all of these cases, references were dated. Therefore, traditional methods of ligand-binding are unlikely to be used in future studies.

Method validation versus publication

Overall, the reporting of key validation data varied indicating that publications are not consistent to support their methods. It appeared as though the reporting of precision and accuracy of the methods was >50%. While this is encouraging and should facilitate the transfer of these validated methods to new applications, it was sometimes unclear as to whether data were derived from validation in plasma matrices or in the matrix of interest. Therefore, some published data may misrepresent the true precision and accuracy of the method as it pertains to the application for analysis of tissues or biofluids.

Consistent with specimen collection and handling, the reported stability of drugs and metabolites in processed and/or stored samples was low (<10%) and offered little support for storage of tissue and biofluids collected during current clinical studies. This is concerning since most of these specimens are analyzed after all have been collected and specimens may be stored for a year or more. Therefore, there is a need for generation of data to support the valid use of clinical samples under various conditions of exposure.

Special considerations for biofluids & tissues

Normalizing tissue and biofluid concentration values may help to provide better modeling outcomes when considering PK and PD relationships. Very few methods reported using such procedures, but the actual application of results was not known. Since these specimens may vary in content and chemical and/or physiological properties or have the potential to be easily contaminated, more guidance should be sought. Biofluids such as saliva, semen and CVF, may vary in viscosity, pH and cellular debris, as well as contain environmental contaminates such as food, cells or blood. Biofluids from the female genital tract may also vary as they are subject to menstrual cycle changes. Breast milk contents vary throughout the feeding time with the fat content increasing toward the end of breastfeeding; the nutritional status of the mother may also cause variation of breast milk contents. Tissues vary in vascular content and their disease states may alter their capacity to function effectively. Even biofluids such as CSF can vary in protein content effectively causing greater variation in drug measurements [11]. Little detail has been provided for normalizing concentration results by appropriate parameters of the specimen. As researchers collect and analyze these uncommon specimens, much information is needed to maintain best practices and include quality assurance.

Current ARV method data are scarce

Most of the references provided information for older ARV, and the current ARV biofluid and tissue method data in newer areas, such as HIV prevention research, are scarce. For example, the use of tenofovir and emtricitabine to prevent HIV transmission will generate a demand for the measurement of these compounds and their metabolites with precision and accuracy at lower limits of concentrations in various biofluids and tissues. The literature searches were completed in 2011 and at that time very few bioanalytical methods had been published for the measurement of tenofovir in biofluids and tissues of interest. Even fewer had been published for measurement of emtricitabine as well as other new ARVs. In the past year, more data have been published, but the details of collection, stabilization, stability, and assay precision and accuracy are lacking [215–228]. There is an important need or gap for data from bioanalytical validations for current ARVs used for treatment and/or prevention. The publishing of such information would serve to expedite the research processes.

Future perspective

Nontraditional specimen types, other than those commonly required for bioanalysis, introduce challenges for the collection, storage, processing and bioanalysis of their contents. As ARV research expands to include treatment of HIV and co-infections, prevention strategies, nanotechnology formulations and eradication from reservoirs, specimens of utmost interest will include many tissue types such as epithelial, connective, nerve, muscle and adipose tissues as well as biofluids such as saliva, breast milk, genital tract samples and CSF. Published ARV method validations for the bioanalysis of tissue and biofluid specimens are available. However, key information, such as specimen collection and handling and ARV stability, is limited and confidence in the reliability of historical published data should be contemplated. Given the importance of reducing the rate of transmission, the goal of eradicating HIV, as well as global economic impact, research in this area would greatly benefit from validated bioanalytical assay reports to facilitate quality, standardized procedures for specimens.

Executive summary.

• ■Standardized specimen collection, handling and storage for biofluids and tissue to be analyzed for antiretrovirals (ARVs) is warranted. Due to the nature of these specimens, stability from collection to analysis for each ARV of interest is needed. Intra-individual variation ought to be characterized.
• ■Bioanalytical methods used to quantitate ARV in biofluids and tissues should be fully validated if concentration results are to be applied as outcomes or to future research.
• ■Characterization of results should be strongly considered in order to reduce or explain some of the variation seen in data within and between individuals and/or specimen types.
• ■Publications Reporting Arv Concentrations Should Provide Key Information For The Robustness Of The Collections, Handling And Storage Of Specimens, The BioanaLysis Method(S) And The Interpretation Of The Concentration Data.
• ■All of these recommendations underscore the importance of sharing scientific information to advance the decline and ultimate termination of HIV infections more rapidly.

Key Terms

HIV treatment and adherence

Patient adherence is critical to success of HIV replication reduction and the measurement of antiretroviral in hair has been shown to correlate with long-term adherence.

HIV eradication

As formulations of drugs begin to target organ specific sites and HIV sanctuaries, the concentration of drug in tissues (e.g., gut-associated lymphoid tissue, brain tissue) becomes more relevant than that in central compartments.

HIV maternal-to-child transmission

As the number of patients receiving antiretroviral as therapy or prevention increases, more unborn children require protection from acquiring HIV. The knowledge of transmission of antiretrovirals through amniotic fluid and placental tissue to the fetal tissues during pregnancy, as well as postpartum through breast milk, is important to obtain.

HIV prevention research

Use of oral and topical antiretrovirals to prevent HIV transmission, as well as treatment as prevention of transmission, has precipitated the collection of many genital compartment specimens.