Quantification of vitamin A and E by LC-MS/MS: a narrative review of the total analytical process

Introduction

Fat-soluble vitamins are a group of vitamins comprised of vitamins A, D, E, and K. As per their name, fat-soluble vitamins dissolve in fat and are stored within the body’s adipose tissue and liver (1,2). In contrast to water soluble vitamins, which are excreted relatively quickly, fat-soluble vitamins accumulate over time to allow the body to maintain reserves, making excessive intake potentially harmful. Of the fat-soluble vitamins, vitamin A and E are essential and cannot be endogenously synthesized, but only obtained through diet.

Vitamin A encompasses retinyl esters, retinaldehyde, retinoic acid and retinol (1). Vitamin A metabolism is complex and multiple genes are involved in conversion of vitamin A before it is utilized by the body. Vitamin A is an essential vitamin with sources obtained from animal or plant origin. Animal sources of vitamin A are found as retinol and its close derivatives, and vitamin A from plants is found as provitamin A (2). Vitamin A is absorbed and metabolized within the proximal portion of the small intestine (2). Dietary retinol is directly absorbed from the intestinal lumen into the enterocyte, whereas dietary retinyl esters are first enzymatically hydrolyzed in the intestinal lumen to retinol prior to entry into the enterocyte (2). Provitamin A carotenoids, synthesized by plants and microorganisms, are enzymatically converted into retinoid or packaged unmodified into chylomicrons and circulate in the body (1,2).

Vitamin A has an important role in vision, and the most significant symptom of acute vitamin A deficiency is vision impairment. If vitamin A deficiency is prolonged and becomes chronic, hyperkeratinization of the ocular tissue occurs, eventually resulting in blindness (3,4). Vitamin A deficiency is a common occurrence in the developing and resource-poor regions of the world due to inadequate nutrition and food deprivation (4). In contrast, malabsorption syndromes such as inflammatory bowel disease are the typical cause of vitamin A deficiency in developed countries (4). Vitamin A intake recommendations vary across populations, with notably higher requirements for pregnant and lactating females (5).

Vitamin A toxicity, or hypervitaminosis, is a rare occurrence but could occur via excessive vitamin A exposure via oral or topical routes. Hypervitaminosis poses significant health risks due to the fat-soluble nature of vitamin A and its tendency to accumulate in body tissues, particularly the liver (3,6). Acute and chronic vitamin A toxicity may lead to a spectrum of adverse effects, including hair loss, dry skin, cracked lips, weakened bones, headaches, and elevated intracranial pressure (6).

Vitamin E is an umbrella term for 8 forms: the tocopherols (alpha, beta, gamma, delta) and tocotrienols (alpha, beta, gamma, and delta) (7), with alpha-tocopherol being the only bioactive form retained in humans (5). Humans are reliant on dietary sources for vitamin E with vegetable oils and nuts comprising the richest source of vitamin E (8,9). Vitamin E binds to free radicals and has important antioxidant properties. Its peroxyl radical-scavenging activities provide protection to cell membranes and limit free radical induced cell damage (1,9).

Vitamin E plays an important immunomodulatory role since deficiency could impair T-cell-mediated functions, increasing susceptibility to infections (9). Vitamin E deficiency is rare, and is typically only observed with fat malabsorption syndromes, such as cystic fibrosis, or specific genetic disorders (10). Clinically, deficiency presents as neuromuscular dysfunction, including ataxia and peripheral neuropathy, as the lack of antioxidant protection leads to oxidative damage in nerve tissues (11). On the other hand, excessive intake of vitamin E may lead to impaired blood coagulation, increased cardiovascular disease risk and increased prostate cancer risk and mortality (12). Another concern with excessive vitamin E intake is its interference with vitamin K metabolism, which could inhibit platelet aggregation and increase the risk of hemorrhaging (12).

Quantification of vitamin A and E concentrations in human matrices is crucial for evaluating nutritional status, diagnosing deficiencies or toxicities, and monitoring the effectiveness of clinical or dietary interventions. Mass spectrometry, especially when coupled with liquid chromatography (LC-MS/MS), is increasingly recognized as a powerful tool for quantifying vitamin A and E due to its increased sensitivity, specificity, multiplexing capabilities, and robust performance in complex matrices (Figure 1). Developing an LC-MS/MS method for vitamins A and E presents several technical and analytical challenges due to the chemical properties of these fat-soluble analytes and the complexity of biological matrices. In this narrative review, we provide an overview of the methodological considerations involved in developing LC-MS/MS assays for vitamins A and E (Figure 2). We summarize available options across the preanalytical, analytical, and quantitative phases. Lastly, we propose a simplified conceptual framework and decision making guide intended to support laboratories in selecting appropriate strategies for the development and optimization of LC-MS/MS methods for vitamins A and E (Figure 3). We present this article in accordance with the Narrative Review reporting checklist (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-31/rc).

Figure 1 Vitamin A and E publications between 1971 and 2025. There has been a significant increase in publications citing mass spectrometric methodology in vitamin A and E quantification. HPLC-UV/ECD, high performance liquid chromatography-ultraviolet/electrochemical detection.

Figure 2 Total analytical process of vitamin A and E LC-MS/MS methodology. The total analytical process of vitamin A and E LC-MS/MS methodology can be divided into pre-analytical, analytical and quantitative phases and there are a multitude of options available within each phase. LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Figure 3 Simplistic decision-making guide for developing vitamin A and E LC-MS/MS method. Each phase of the total analytical process is interdependent and a decision within one phase inadvertently affects the next phase. A simplistic decision-making guide is presented to demonstrate the interconnectivity of each phase of the total analytical process. LC-MS/MS, liquid chromatography-tandem mass spectrometry; PFP, pentafluorophenyl.

Methods

This narrative literature review was conducted by searching PubMed, Google Scholar and Scopus. This narrative review is divided into three sections: preanalytical, analytical and quantitative. Analytical-related content was retrieved from peer-reviewed journal articles published within 2015–2025 using the search terms “vitamin A”, “vitamin E”, “mass spectrometry”, “total analytical process”. An additional search of published peer-reviewed articles from 2015 onwards, using combination of search terms “sample stability”, “sample preparation” was performed to include clinical, preanalytical and quantitative content. A detailed search strategy is summarized in Table 1.

Pre-analytical

Choosing specimen matrix for vitamin A and E analysis

The choice of sample matrix for vitamin A and E analysis is dependent on ease of collection, analyte concentration, sample stability and clinical utility. Whole blood, serum, plasma and urine are the most common specimen types in the clinical laboratory since collection is less invasive, and biomarkers are typically stable and abundant in these matrices.

Plasma and serum

Vitamin A and E are found in micromolar concentrations in plasma and serum, and they are typically the matrix of choice for assessing vitamin A and E nutritional status. However, careful consideration must be taken for storage and sample stability since these vitamins are reported to be labile to light, heat, oxidation, and low pH conditions.

Clinical laboratories can review published literature and best practice recommendations to determine appropriate vitamin A and E sample storage and transportation (13). In general, it is recommended that vitamin A samples are protected from light with aluminum foil or amber tubes (13,14). Vitamin E is not considered photosensitive, but since most methods multiplex vitamin A and E, vitamin E handling typically follows vitamin A storage and transportation requirements. Given the lack of harmonization in sample preparation, study design and sample stability acceptance criteria, there are conflicting findings for vitamin A and E stability at room temperature (RT), refrigerated (4 ℃) or at −20 ℃. For example, published literature cites vitamin A and E sample RT stability ranging from 24 hours to 1 week (13-15). Guidelines and multiple studies provide support that optimal long-term storage of vitamin A and E should be at −20 ℃ (13). However, despite the availability of published literature and best practice recommendations, it is suggested that laboratories perform their own stability studies to assess storage and transportation conditions that reflect the laboratory’s workflow process.

Whole blood

Whole blood provides a stable alternative matrix source for assessing vitamin A and E nutritional status (16,17). However, since both vitamin A and E are fat-soluble, there is a negligible amount of vitamin A and E within red blood cells. Vitamin A circulates by binding to retinol-binding protein, and vitamin E often circulates by binding to lipoproteins. Measurement of vitamin A and E in whole blood requires more complex sample preparation technique and additional clean-up of the sample prior to LC-MS/MS analysis. Furthermore, the addition of other cellular components (red blood cells, white blood cells, and platelets) in whole blood would dilute the concentration of measured vitamin A and E. Vitamin A and E are not particularly more stable in whole blood relative to serum and plasma (15). Lastly, the World Health Organization (WHO) guidance for vitamin A deficiency is based on serum (18,19). Thus, the use of whole blood makes it more difficult to interpret nutritional status against clinical guidelines.

Urine

Urine is typically not a reliable matrix for assessing vitamin A and E nutrition status, because both are fat-soluble vitamins and are not excreted in meaningful amounts in urine. Vitamin A circulates in the body bound to retinol binding protein (RBP), and recent studies identified the clinical utility of RBP as an indicator of kidney function (20,21). However, solid-phase extraction (SPE) columns typically used for retinol sample preparation can result in significant loss of creatinine. Creatinine is a highly polar molecule and would not be retained by the non-polar stationary columns used for retinol extraction. Clinical interpretation is difficult if one cannot standardize urinary RBP to urinary creatinine. Although a chromatographic method capable of multiplexing urinary RBP and urinary creatinine has been described in the literature, it has not been widely adopted in routine clinical laboratory practice (20).

Dried blood spot (DBS)

DBS specimens offer minimally invasive, low-cost, and easily transportable sampling. Despite these advantages, quantifying retinol from DBS has long posed challenges due to concerns with analyte stability, storage, sample preparation, recovery, and internal standard (IS) selection. Published literature demonstrated that antioxidant-treated paper and vacuum treatment could improve DBS retinol stability significantly for up to 30 days (22). However, accurate quantification of retinol in DBS requires optimization of DBS formation, analyte release and IS selection (23).

Sample preparation techniques for measurement of vitamins A and E in clinical analysis

Vitamins A and E are small, lipophilic molecules bound to blood proteins, requiring dissociation for accurate measurement. Moreover, sample preparation impacts data quality of LC-MS/MS assays, as this methodology is more susceptible to interferences, such as phospholipids originating from the matrix, or from other chemicals and plasticizers from test tubes. These interferences can adhere to the stationary phase of chromatographic columns and deposit in the ionization source, resulting in either ion suppression or ion enhancement, directly impacting the sensitivity, specificity, and accuracy of the assay (15,24). This impact can be more profound depending on the type of mass spectrometer and ionization source used (24).

For clinical applications, the choice of sample preparation process depends on many factors, such as the biological matrix, chemical properties of the analytes, cost of analysis per sample, and turnaround time. The most common sample preparation techniques are protein precipitation (PP), liquid-liquid extraction (LLE), and SPE often used alongside other purification processes such as filtration and centrifugation (Table 2). Regardless of the sample preparation technique, it is strongly recommended to incorporate the use of appropriate IS to ensure accurate quantitation of analytes, to correct for potential matrix effect, and to control the recovery or loss of analytes during any step of the analytical process. IS are added to samples during the early steps of the analytical process and undergo the same treatment as the sample. The response ratio of the analyte to the IS is known as the area ratio and is used to provide accurate and precise quantitation results.

PP

PP is the fastest clean-up method for vitamin A and E analysis and helps free these vitamins from their protein carriers. The choice of organic solvent requires it to be miscible with the sample and the mobile phase. The solvents commonly used for PP are ethanol (EtOH) (24,31,43,49,55,68), methanol (MeOH) (32,35,42,47,48,58-61), acetonitrile (ACN) (20,24,37,38,54), and isopropanol (IPA) (42,50,64), or a combination of these. EtOH, MeOH, and ACN are commonly found in clinical laboratories and are inexpensive, making them the solvent of choice for PP. Although PP is a quick and inexpensive sample preparation option, it often results in poor analyte recovery, insufficient protein removal, and ion suppression due to the presence of phospholipids in the eluents. Additional sample cleanup techniques are required after PP to minimize ion suppression.

LLE

LLE is based on the partitioning of an analyte between two immiscible liquid layers, usually water and a water-immiscible organic solvent. LLE does not require additional automation equipment, making it more cost-effective and the preferred sample preparation technique for many clinical laboratories. The organic solvents commonly used for LLE are hexane (10,30,32,35,38,42,43,48,54,56,58,59,61,63), n-hexane (60,62), isooctane/chloroform (29,68), or IPA/heptane (64). Chloroform is considered carcinogenic and is typically not the solvent of choice (48,49). In LLE, analytes are recovered from the organic layer and further concentrated by decreasing the eluent volume through evaporation under nitrogen flow. LLE is a simple technique and uses basic laboratory equipment, but requires large sample volume and organic solvents, leading to prolonged evaporation time, especially when multiple extractions of the samples and a combination of eluents are required (56,58,60,64).

Supported liquid extraction (SLE)

SLE is an alternative form of LLE. SLE also exploits the partitioning properties of analytes between two phases. Unlike LLE, SLE utilizes packed columns or cartridges of diatomaceous earth to partition analytes from impurities (27,30,64). It is noteworthy that phospholipids cannot be removed with SLE. If phospholipids are undesirable in the sample matrix, additional sample preparation techniques are required. The sample is usually diluted before being loaded onto the diatomaceous earth, and the analytes are eluted with the same solvents that are commonly used for LLE extraction. SLE provides added benefits over LLE as it has smaller sample volume requirements, less consumption of organic solvents and can be performed in a 96-well plate format, making it amenable to automation.

SPE

SPE is another sample preparation technique that can be used on its own or in conjunction with PP (21,34,45,49,69). When samples are loaded onto an SPE plate, analytes are retained within the SPE material, and washing steps will allow impurities to pass through. The analyte from the SPE plate is then eluted and collected with an elution solution. Wash buffer and elution reagent composition will vary based on the SPE material. SPE leads to cleaner eluents but requires more steps than SLE. Similar to SLE, SPE is readily automated and can be integrated into an online workflow, with online SPE performed directly on the liquid chromatography (LC) system. In online SPE, the SPE cartridge is installed before the analytical column, then a switching valve allows either the entire or partial eluate from the SPE to proceed to chromatographic analysis (34). SPE is more efficient than LLE due to smaller sample volume requirements (49).

Analytical

Liquid chromatography separation for fat-soluble vitamin analysis

Due to increased sensitivity, wider dynamic range and better separation of analytes, LC coupled with spectrophotometric detection or mass spectrometry is now the most widely used approach for vitamin analysis. Since vitamin A and E lack ionization sites, selection of appropriate chromatographic columns and proper optimization of mobile phase composition are of utmost importance in making vitamin A and E amenable to LC-MS/MS analysis.

Column selection

Column dimensions, particle size and chemistry of the packing material have direct impacts on retention and resolution of analytes. Long columns (100–150 mm) suffer from high back pressure and require high pressure LC pumps (UHPLC). Differing column diameters impact mobile phase flow rates. Mobile phase flow rate plays an important role in the ionization efficiency, which ultimately determines the sensitivity of the assay. Columns with internal diameters of 3.0–4.6 mm have longer retention times and require higher mobile phase flow rates (usually greater than 1 mL/min) to ensure maintenance of linear velocity. The introduction of 2.1 mm diameter columns resulted in methods with shorter analysis time, slower flow rates, less solvent consumption and significant sensitivity improvements (70).

In terms of stationary phase chemistry and separation mechanism, reverse-phase chromatography (RP-HPLC) is the most extensively used separation technique for vitamin A and E analysis, due to its compatibility with mass spectrometry detection, reproducibility of retention times, fast equilibration times, good peak shape and robustness. The chromatographic separation on RP-HPLC is based on the hydrophobicity of analytes and their interaction with a non-polar stationary phase, usually an alkyl-based stationary phase (C8, C18, C30), and a polar mobile phase, usually a mixture of water and organic solvents.

The choice of a stationary phase column for vitamin A and E quantification is dependent on whether vitamer separation is required. C18 columns are frequently used for vitamin A and E quantification (51,52). However, C18 columns with high carbon load cannot provide the adequate resolution required for separation of structurally similar vitamers and therefore cannot be used to separate the isobaric compounds, beta and gamma tocopherol. C30 columns are also known as “carotenoid” columns (62). These columns are more hydrophobic than C18 columns and provide sufficient phase thickness to enhance interactions with long-chain molecules, enabling the separation of geometric and positional isomers of conjugated double-bonding systems. Despite their efficiency in the separation of carotenoids, C30 columns result in broader chromatographic peaks and partial separation of beta- and gamma-tocopherol.

When resolution of structurally similar vitamin isomers is required, non-alkyl columns can be considered as an alternative. These stationary phase chemistries enhance separation of structurally similar vitamers by leveraging alternative mechanisms of interactions, such as π-π, hydrogen bonding, dipole-dipole and steric interactions. Some examples of non-alkyl columns that have been reported for vitamin A and E analysis by liquid chromatography are pentafluorophenyl (PFP) (15,20,28,35,37,47,53,63), pentafluorophenylpropyl (F5) (24,30,61), phenyl (22,38,43,48), biphenyl (27) and phenyl-hexyl (49). Most of these studies focus on the quantification of vitamin A and alpha-tocopherol either as single analytes or as part of multiplex panels for fat-soluble vitamins quantification, including 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, epi-25-hydroxy vitamin D3 and vitamin K.

Mobile phase selection

The selection of mobile phase composition is an equally important factor requiring optimization during method development. Optimization of the mobile phase composition includes choosing an optimal organic solvent, organic additives, and optimal pH. Organic solvents in the mobile phase significantly impact the interaction of analytes with the stationary phase and directly impact analyte retention time. MeOH and ACN are commonly used as organic solvents due to their compatibility with mass spectrometry. Volatile organic additives are added to the mobile phase to enhance analyte ionization, and improve chromatographic characteristics such as peak shape and chromatographic resolution. Examples of organic additives include formic acid, ammonium acetate, ammonium formate and ammonium fluoride. Since vitamins A and E are pH labile, the pH of the mobile phase should not significantly deviate from neutrality. Formic acid can be used alone or in conjunction with ammonium salts in mobile phase buffers. The addition of ammonium salts increases the ionic strength of the mobile phase, while ammonium ions act as a weak base, facilitating buffering of the pH from formic acid. It has been reported that combining ammonium acetate and acetic acid in the mobile phase increased sensitivity of fat-soluble vitamin quantification compared to ammonium acetate alone (62). Common formic acid concentration in the mobile phase is 0.1%, v/v, and ammonium acetate and ammonium formate concentration varies from 2 to 100 mmol/L (26,55).

Mass spectrometric detection of vitamin A and E: ionization modes, reported transitions and IS considerations

Chromatography is a separation technique and is typically paired with a detection system for vitamin quantification. Several detection systems have been previously described for vitamin A and vitamin E analysis. Many studies have reported the use of UV-Vis detection, but they usually suffer from poor sensitivity and selectivity. Fluorometric detection (FLD) has also been reported for the quantification of vitamin A and E. However, mass spectrometry has gained a lot of interest due to its superior sensitivity and specificity compared to spectroscopic detectors.

Ionization source and mass spectrometer selection

Mass spectrometry confirms and quantifies the presence of a compound based on its molecular structure information by separating ions based on their mass-to-charge ratio (m/z) and measuring their abundance. The selection of the ionization source and mass spectrometer instrumentation should be based on the requirements of an assay. However, it appears that cost and availability are typically the two main driving forces for the selection of instrumentation, especially in clinical laboratories.

Sensitivity of an LC-MS/MS method is dependent on ionization efficiency. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are the most frequently used ionization techniques for vitamin analysis in clinical settings and research laboratories. Ionization source selection is primarily dictated by cost and instrument availability, as most mass spectrometers are equipped with ESI, and many laboratories lack the resources to acquire an APCI source. Although ionization occurs later in the analytical process, it should be one of the first considerations when deciding on preceding steps. Understanding the advantages and limitations of each ionization source is essential, as this knowledge informs decisions throughout all preceding stages of the analytical workflow. APCI has been historically reported to provide higher sensitivity due to the greater ability to ionize non-polar compounds and, relative to ESI, is less susceptible to matrix effect (71,72). For instance, PP extracts contain high concentrations of phospholipids that, when analyzed using ESI, can lead to substantial ion suppression. In such cases, APCI—which is inherently less affected by phospholipid mediated matrix effects—represents a more suitable ionization source for PP samples (Figure 3). While ESI is highly susceptible to matrix effect, it has been reported to provide higher responses for fat-soluble vitamins when compared to APCI (31,49,54). If ESI is the only available option, a more rigorous chromatographic separation, such as an extended or steeper gradient, can be employed to improve analyte-matrix resolution and mitigate suppression.

In positive ionization, both ESI and APCI lead mainly to the formation of [M+H-H₂O]⁺ ions for vitamin A with m/z 269, and [M+H]⁺ ions for alpha tocopherol with m/z 431. The [M+H]⁺ ions may subsequently undergo hydrogen dissociation, forming [M-H]⁺ molecular ions with m/z 429. Previous studies have demonstrated that these two molecular ions consistently co-occur in a fixed ratio, and that [M]⁺ molecular ions are present for certain vitamin E isoforms when aprotic solvents (such as ACN) replace methanol in the mobile phase (70). Alpha-tocopherol analysis using negative ionization on APCI has been reported, using the [M-H]^- molecular ion with m/z 429.4 (30,65).

In terms of mass spectrometers, the triple quadrupole is the most frequently used type of mass spectrometer for vitamins A and E analysis due to its analytical sensitivity, affordability and targeted analysis capabilities (15,20,22-24,26-28,30-33,35,38,41,44,47,49-53,55-57,61,63,64,66,73). In a triple quadrupole, the precursor ions of interest are selected in the first quadrupole (Q1), fragmented in a collision cell, and specific product ions are selected and detected in the third quadrupole (Q3). Because the fragmentation and precursor-to-product ion transitions are precisely controlled, tandem mass spectrometry allows for highly selective and sensitive detection of target analytes in the presence of complex sample matrices. Data acquisition is termed Selected Reaction Monitoring (SRM) when one transition per parent ion is monitored, and Multiple Reaction Monitoring (MRM) when two or more transitions are used. The most abundant fragment ion is usually used for analyte quantification and is known as the “quantifier ion”, whereas the “qualifier ion” is monitored to verify analyte identification and ensure that the observed peak is not an interference. Commonly reported fragments are 93 m/z and 119 m/z for vitamin A and 165 m/z and 137 m/z for vitamin E (30).

Quantification

IS are essential in mass spectrometric analysis of vitamins A and E as they compensate for matrix effects and enable absolute quantification. IS are structurally similar, non-endogenous compounds with distinct m/z ratios. They are added at the initial steps of sample preparation to compensate for variations that can occur during sample preparation, injection, and ionization. Cost, stability requirements, and commercial availability are key points that drive the selection of IS. When exact matches are unavailable, closely eluting analogs may be used (13). Both deuterated and ¹³C isotope labelled IS, are widely used for LC-MS/MS analysis of vitamins A and E. Due to slight hydrophobicity differences, deuterated IS elute slightly earlier than their corresponding analytes. ¹³C-labelled IS have the same physical and chemical characteristics as their corresponding analytes, resulting in almost identical chromatographic behavior and co-elution.

Calibration approaches for vitamin A and E analysis in mass spectrometry: challenges and current practices

CLSI guidelines recommend six-point calibration curves, including a blank, for mass spectrometry-based methods. However, commercial serum calibrators for vitamins A and E have only recently become available and remain underutilized. Commutability of calibrators is another important consideration in mass spectrometry to ensure accurate results and minimize bias due to matrix differences between the calibrators and patient samples. Previous studies demonstrated poor commutability between commercially available calibrators (74), likely attributed to traceability differences and raising concerns about lack of harmonization, and poor inter-laboratory comparability. In-house matrix-matched calibrators are commonly used but challenging for vitamins A and E due to their endogenous presence and lack of vitamin-free serum matrices.

Based on the studied literature, different approaches have been adopted for calibration. Most studies reported the use of neat organic solvents, either EtOH (41,43,48,55,64,65,73,75) or MeOH (26,42,47,49,56,58-60,66,69) as blanks for calibration curve preparation. Stripped matrices, filtered through an activated carbon filter to remove non-polar/lipophilic components, have been frequently reported for calibrator preparation (20,21,23,27,30,35-37,53). Surrogate or synthetic matrices like bovine serum albumin (BSA) in phosphate buffered saline (PBS) and sodium chloride solution (NaCl) are another frequently used blank alternative (22,24,28,31,34,38,54,62). Three studies demonstrated use of the background subtraction approach (29,61,68), where the calibration curve is prepared by spiking pooled matrices and subtracting endogenous analyte levels.

Harmonization and standardization of vitamin A and E methods in clinical settings: challenges and current practices

Harmonization of methods is a crucial aspect in clinical biochemistry. Although there are many similarities between the published vitamin A and E methods, there are many factors impeding standardization and harmonization of these assays. The labile nature of vitamin A and E makes it challenging to create a stable certified reference material (CRM) with a multi-year shelf life. In fact, the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database does not list a reference measurement procedure or reference material for serum vitamin A and E. This narrative review identified multiple variables for developing a vitamin A and E LC-MS/MS method. It would be difficult to create a CRM commutable across all methods when there are many variables involved. An ideal characteristic of a CRM is commutability across all methods. The lack of a reference measurement procedure and low clinical demand for vitamin A and E quantification contribute to delayed development of vitamin A and E CRM.

Although vitamin A and E CRM is not yet available, efforts by the Australasian Association of Clinical Biochemists (AACB) Vitamins Working Party and the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) created evidence-based guidelines to promote fat-soluble vitamin method harmonization. They identified critical components that affect vitamin A and E analysis in blood and serum and highlighted recommended best practices to promote harmonization between labs (13).

Limitations

Although we aimed to provide a broad overview of available approaches, numerous additional methodological options for each aspect of the total analytical process exist that were not included in this narrative review. Our narrative review focused specifically on the timeframe between 2015–2025. With the fast rate of method development, additional published literature may not have been captured by our review. Previous systematic reviews have examined analytical approaches for fat-soluble vitamins more broadly, including methods for quantifying and multiplexing multiple analytes within one method (70,76). In contrast, our review focused specifically on vitamins A and E and therefore has a more limited scope relative to those comprehensive evaluations. Because our objective was to perform a narrative review and not a systematic review, the decision-making guide (Figure 3) presented here is intentionally simplified. We acknowledge that many other factors must be considered when developing an LC-MS/MS method. Our intention was to offer readers a practical introductory framework rather than an exhaustive methodological roadmap. Developing a more exhaustive or universally applicable framework would require a systematic review, which future studies may undertake.

Conclusions

Mass spectrometry-based methods have emerged as the superior approach for quantification due to increased sensitivity and specificity. The choice of sample preparation techniques, chromatographic separation methods, and ionization sources plays a crucial role in method development. Figure 2 summarizes the range of methodological options presented in this narrative review. LLE remains the most widely used sample preparation technique (Table 2), while reverse-phase liquid chromatography is the preferred separation method. ESI has become the most common ionization technique, although APCI still has its proponents. Figure 3 reiterates the interdependency of each stage of method development, and choices made inevitably influence subsequent steps. Together, these developments underscore the continued evolution of LC-MS/MS-based vitamin analysis and highlight the importance of selecting method components in a systematic and integrated manner.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Sukhbinda Kaur) for the series “Trace Elements and Vitamins” published in Journal of Laboratory and Precision Medicine. The article has undergone external peer review.

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-31/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-31/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-31/coif). The series “Trace Elements and Vitamins” was commissioned by the editorial office without any funding or sponsorship. T.K., Y.W. and D.T. are employees of LifeLabs. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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