Vitamin K refers to a group of fat-soluble vitamins best known for their role in blood coagulation. Other biological processes that Vitamin K has been implicated include blood calcium regulation, vascular anti-calcification, and bone metabolism (1). There are two main forms of Vitamin K; Vitamin K₁ (phylloquinone; PK) and Vitamin K₂ (menaquinones; MK). Phylloquinone is most abundant in green leafy vegetables and their oils, but is also present in smaller concentrations in the majority of food groups such as fruit, meat, and dairy products (2). In contrast to PK, the MKs are a group of isoprenologs where the side chain varies by the number of isoprenoid units ranging from four to thirteen repeats (MK4 to MK13; see Supplementary Figure 1) (3). Despite having analogous structures, the origins of menaquinones differ. MK4 is synthesized from PK by animals, and thus is obtained in the diet from animal products (3). All other MK are synthesized by anaerobic bacteria and thus are found in fermented foods, such as cheese (4).

Presently, there are several databases listing the PK content in a range of foods, especially vegetables and fruits. The most extensive of these databases has been developed by the United States Department of Agriculture (USDA) that has recently been updated (5). The USDA National Nutrient Database has recently been combined with the United Kingdom Composition of Foods Integrated Dataset (COFID) to provide an update to Vitamin K values in the Irish Food Composition Database (6). Although MK4 content of some food are listed, the USDA database does not include other MKs (MK5-12). Generally, such MKs are found in much lower quantities in meats and cheese compared to MK4, with the exception of MK7 known to be high in fermented foods such as Natto (7). Besides the USDA, MK reference ranges for food products are also limited to a few individual investigations (4, 7–10).

There are numerous methods to measure both PK and MK in biological matrices, as reviewed previously (11, 12). However, there is presently no standardized method to quantitate PK and MK in food. This has likely contributed toward a lack of detailed information on the Vitamin K content of food, which is reported to vary by regions including Europe, Asia and the United States of America by up to 50% (13). In Australia, a Vitamin K database for commonly consumed foods does not exist. This can limit nutritional epidemiology when researchers seek to investigate the potential health benefits of Vitamin K (PK and/or MK) for a range of health outcomes in Australian cohorts. Since MKs are estimated to constitute about 10% of total dietary Vitamin K intake, with the majority (up to 40%) of dietary MK attributed to MK4 (3, 14), the aim of this investigation was to develop a preliminary Vitamin K database, assessing PK and MK4 for commonly consumed commercially available foods in Australia.

For the first time, we present an Australian food composition database for Vitamin K. Specifically, we present the PK, MK4 and MK7 content of 60 commonly consumed commercially available foods. Food items analyzed comprised a wide range of food groups known to provide the majority of dietary Vitamin K1 and K2, including vegetables, oils, meat, and dairy. We were able to accomplish this by adopting an LCMS/MS method to assess the Vitamin K content of these food items. Findings of this work may help with the revaluation of Vitamin K intake guidelines in Australia where an adequate intake of 60 and 70 μg/day for men and women, respectively, is proposed (23), which are substantially lower than the USA (120 μg/day for men, 90 μg/day for women) (24).

Similar to previous work, vegetables were found to be a major source of PK. We report that specific vegetables including spinach and kale as the richest sources of PK (263, 129 μg/100 g, respectively). Noteworthy, the PK content of these vegetables differed substantially compared to previous work. For example, Schurgers and Vermeer report that PK in spinach and kale to be 387 and 817 μg/100 g. Alternatively, the USDA report these measurements to be 483 and 390 μg/100 g, respectively. Compared to these studies, PK content in spinach we measured varied between 33 and 45%. For kale, the PK content was approximately 6 times lower compared to Schurgers and Vermeer (8), and 3 times lower compared to the USDA. Nevertheless, it has been suggested that the average PK content in “green vegetables” to be in the range of 100–750 μg/100 g (14). Although canola oil and margarine were shown to contain a high-moderate amount of PK, these foods are typically consumed in quantities less than 100 g/day. Therefore, such foods may not be a large contributor toward overall dietary Vitamin K intake. Collectively, these results highlight that the importance of accounting for regional differences in PK content of food to reduce inaccuracies when trying to estimate PK intake for specific populations. Noteworthy, the Vitamin K content of fruits are reported to be low (25, 26), a finding we also observed in apples and pears in this study. Although it would have been ideal to assess the Vitamin K content of more fruits, due to limited resources, we selected some of the most commonly consumed fruits previously shown to contain Vitamin K.

Others including ourselves have now demonstrated that the MK4 in foods also differ between regions (13). Specifically, MK4 content of beef cuts from the USA (1.1–9.3 μg/100 g) was substantially lower compared to Japan (15.0 ± 7.0 μg/100 g) (9, 18). Egg yolk MK4 content was also 4 times greater in Japan compared to the USA (64 vs. 15.5 μg/100 g, respectively). In comparison, our measurements in whole egg and beef indicated a MK4 content of 33 and 16 μg/100 g, respectively. Such regional differences may be attributed to differences in food production including the use of menadione in animal feeds (13). Hard and soft cheese are also known to be dietary sources of MKs (4, 13) that provide between 4 and 10 μg/100 g (13). These findings are consistent with our results where MK4 content of brie, cheddar and parmesan were 9, 6 and 5 μg/100 g, respectively. Although not assessed, it is worth highlighting that up to 15% of variability has been reported in the MK content (MK-6 to MK-10) of semi-hard cheese varieties from three different European countries, including France, Poland and Denmark (4). Similar to previous work, MK7 was not detected (or found in very small amounts) in cheese varieties (13), whilst margarine was found to contain almost double the amount of MK7 compared to MK4 (6 vs. 11 μg/100 g, respectively). In line with previous work, natto was found to be a rich source of MK7 (82 μg/100 g). However, this was substantially lower compared to the MK7 content previously measured (902–998 μg/100 g) (9, 13, 21).

In order to create food databases, a validated method of measuring PK and MKs simultaneously, with accuracy and high throughput, is required. This is further complicated as the Vitamin K content in foods are typically low, in conjunction with the added complexity of extraction from the food matrix (27). We have previously provided an overview of the current methods for analyzing Vitamin K compounds in food (12). An emerging method to measure Vitamin K in foods is a HPLC-MS method developed by Karl et al. (20), which has high versatility and may be used to measure PK and all MKs in a variety of forms such as food, serum and feces. A combination of HPLC and gas chromatography-MS (GC-MS) has been used with deuterium-labeled internal standards to accurately measure PK in serum (28). However, there are limitations in using GC for measuring Vitamin K, as high temperatures are needed to volatise Vitamin K compounds (>300°C) (29). Therefore, HPLC is the preferred method to separate Vitamin K compounds for analysis. Previously, reverse-phase HPLC with fluorescent detection or HPLC–MS, with K1 or deuterated-K1 as the internal standard have been used to determine PK and MK from food (4, 20). Most recently, a whole range of fermented foods were assessed for their PK and MK content using a newly developed ultra-high performance liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometric (UHPLC-APCI-MS/MS) method (21). They reported a LOD and LOQ for PK and MK4 of 1.0, 3.2 pg and 6.4, 21.2 pg, respectively. In the present study, we used HPLC method coupled to heated electrospray ionization tandem mass spectrometry (H-ESI-MS/MS) to measure PK and MK in different foods with an improved chromatographic separation and detection of Vitamin K's and their analogs with the LOD and LOQ for PK and MK4 as 1 and 5 pg (0.1 and 0.5 ng/ml) and 5 and 10 pg (0.5 and 1 ng/ml), respectively. These values are comparable to that reported by Tarvainen et al. (21). In comparison, we have developed an efficient LCMS/MS method with a shorter chromatography run time for the quantification of PK and MK4 with the use of deuterated internal standards. Specifically, when adopting a modified LCMS/MS method here (30), we recorded faster elution of PK and MK4 retention times (1.90 and 1.53 mins), compared to previous work analyzing fermented foods (5.52 and 4.66 mins) (21) as well as human serum and plasma (12.20 and 10.41 mins) (19).

In conclusion, we have provided the Vitamin K (PK, MK4 and MK7) content of commonly consumed commercially available food products in Australia. The LCMS/MS method enabled the quantification of PK, MK4, and MK7 in these food products with acceptable LOD and LOQ and inter and intra-assay precision. In conjunction with other Vitamin K food database, this data can be used by nutritional epidemiologist seeking to quantify dietary intake, especially in Australian cohorts, for a range of health outcomes.

Within reasonable request, the raw data supporting the conclusions of this article will be made available by the authors.

MS, LB, JL, KC, and JH conceived and designed the study. CP and HK performed the analysis. SS and KC did the LCMS/MS method development. CP and MS prepared the manuscript. CP had the primary responsibility for the final content. All authors reviewed, read, and approved the final manuscript.

This research was supported by a Royal Perth Hospital Research Foundation Springboard Grant. The salary of MS is supported by a Royal Perth Hospital Research Foundation Career Advancement Fellowship (CAF00/2020). The salary of LB is supported by a National Health and Medical Research Council (NHMRC) of Australia Emerging Leadership Investigator Grant (ID: 1172987) and a National Heart Foundation of Australia Post-Doctoral Research Fellowship (ID: 102498). The salary of JL is supported by a National Heart Foundation of Australia Future Leader Fellowship (ID: 102817). The salary of JH is supported by a National Health and Medical Research Council of Australia Senior Research Fellowship (ID: 1116973). None of the funding agencies had any role in the conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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